VECTOR-BASED MUTAGENESIS SYSTEM

Strategies, reagents, methods, and systems for modulating the mutation rate in cells are provided herein. The strategies, reagents, methods, and systems are broadly applicable for the modulation of mutation rates in cells where high mutation rates and/or control over a broad range of mutation rates is desired, for example, in the context of diversifying a nucleic acid sequence or a plurality of such sequences within a population of cells, for the generation of diversified nucleic acid libraries, and for directed evolution of nucleic acids and encoded products.

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Description
RELATED APPLICATIONS

The present application claims the benefit under 35 U.S.C. § 119(e) to U.S. provisional patent applications. U.S. Ser. No. 62/149,378, filed Apr. 17, 2015, and U.S. Ser. No. 62/272,035, filed Dec. 28, 2015 the entire contents of each of which are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under grant HR0011-11-2-0003, awarded by Defense Advanced Research Projects Agency (DARPA), and under grant N66001-12-C-4207 awarded by the Space and Naval Warfare Systems Command (SPAWAR). The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The random mutagenesis of DNA provides the genetic diversity that fuels evolution both in nature and the laboratory. Methods to enhance unbiased, random mutagenesis in cells offer major advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency, and narrow mutational spectra.

SUMMARY OF THE INVENTION

Some aspects of this disclosure provide strategies, reagents, compositions (e.g., vectors), kits, methods, and systems for modulating the mutation rate in cells. The strategies, reagents, compositions, kits, methods, and systems are broadly applicable for the modulation of mutation rates in cells in any context in which high mutation rates and/or control over a broad range of mutation rates is desired, for example, in the context of diversifying a nucleic acid sequence or a plurality of such sequences within a population of cells, for the generation of diversified nucleic acid libraries, and for directed evolution of nucleic acids and encoded products. While some uses and applications are described in detail herein, additional suitable uses of the strategies, reagents, compositions, kits, methods, and systems provided herein will be apparent to those of skill in the art based on the present disclosure.

Phage-assisted continuous evolution (PACE) can serve as a rapid, high-throughput method to evolve genes of interest. One advantage of the PACE technology is that both the time and human effort required to evolve a gene of interest are dramatically decreased as compared to conventional iterative evolution methods. The general concept of PACE technology has been described, for example, in International PCT Application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. application, U.S. Ser. No. 13/922,812, filed Jun. 20, 2013; and U.S. application, U.S. Ser. No. 62/067,194, filed Oct. 22, 2014, the entire contents of each of which are incorporated herein by reference. During PACE, a phage vector carrying a gene encoding a gene of interest replicates in a flow of host cells through a fixed-volume vessel (a “lagoon”). For example, in some embodiments of PACE described herein, a population of bacteriophage vectors replicates in a continuous flow of bacterial host cells through the lagoon, wherein the flow rate of the host cells is adjusted so that the average time a host cell remains in the lagoon is shorter than the average time required for host cell division, but longer than the average life cycle of the vector, e.g., shorter than the average M13 bacteriophage life cycle. As a result, the population of vectors replicating in the lagoon can be varied by inducing mutations, and then enriching the population for desired variants by applying selective pressure, while the host cells do not effectively replicate in the lagoon.

The rate at which host cells mutate a gene of interest during a directed evolution experiment can affect the time required to evolve a gene of interest to a desired state, and may even limit the mutations that can be achieved during a standard PACE experiment. While conventional methods of mutagenesis are effective, in some circumstances, higher mutation rates are desirable. Some aspects of this disclosure provide potent, inducible, broad-spectrum, and expression construct-based (vector-based) mutagenesis systems for use in bacterial cells, e.g., E. coli cells. These recombinant expression constructs, also referred to as mutagenesis constructs or, if they are in the form of a plasmid, as mutagenesis plasmids, can enhance mutation rates by over 300,000-fold as compared to basal levels when fully induced, surpassing the mutational efficiency and spectra of the most widely used in vivo and in vitro mutagenesis methods. In some embodiments, the mutation rate can be modulated via inducible expression of mutagenic gene products from the expression constructs provided herein, which are useful for modulating the mutation rates as desired, e.g., during different phases of a directed evolution experiment.

Some aspects of this disclosure demonstrate the usefulness of the mutagenesis systems provided herein in a directed evolution context, e.g., for evolving biomolecules with novel or changed properties. The improved mutation rates are demonstrated, for example, by the data provided in the Examples, showing that novel antibiotic resistance can be evolved in wild-type E. coli in less than 24 hour using the mutagenesis expression constructs, thus outperforming all known methods for inducing mutations in host cells, including, for example, chemical mutagens, UV light, and the mutator strain XL1-Red under similar conditions. The mutagenesis systems provided herein also allowed for the rapid continuous evolution of T7 RNA polymerase variants capable of initiating transcription using the T3 promoter in less than 10 hours without requiring any evolutionary stepping-stones or initial mutational drift, in contrast to previously described mutagenesis systems with lower mutation rates. The mutagenesis systems, methods, and kits provided herein can be applied, for example, to the high-frequency, broad-spectrum mutagenesis of chromosomal, episomal, and viral nucleic acids in vivo, and are applicable, inter alia, to a variety of cells, e.g., bacterial, yeast, and eukaryotic cells, and a variety of applications, e.g., both bacterial or bacteriophage-mediated laboratory evolution platforms. Those of skill in the art will understand that these examples are provided to illustrate some non-limiting, possible applications of the mutagenesis systems and expression constructs provided herein, and that the present disclosure embraces additional applications and is not limit in this respect.

In some embodiments, a plurality of nucleic acid sequences encoding a gene product that increases the mutation rate in a cell are employed, e.g., combinations of such gene products as shown in Table 2 or FIG. 7. In some embodiments, combinations of two or more nucleic acid sequences encoding different gene products that increases the mutation rate in a cell may be included in a single expression construct, e.g., in a multi-cistronic construct in which a single promoter, e.g., an inducible promoter as described herein, drives expression of all or a plurality of the different encoding sequences, or in an expression construct comprising two or more promoters, each driving expression of at least one of the encoding sequences. In some embodiments, a combination of different encoding sequences may be provided on different expression constructs, each of which carrying at least one of the different encoding sequences. Suitable configurations or monocistronic, multicistronic, and multi-vector expression constructs for expression of the combinations of mutagenesis-inducing gene products in cells will be apparent to those of skill in the art in view of the present disclosure.

In some embodiments, the gene product that disrupts a proof-reading pathway is a dnaQ926, BRM1, BR11, BR1, BR6, or BR13 gene product. In some embodiments, the gene product that disrupts a translesion synthesis pathway is an umuD′, umuC, recA, dinB, or polB gene product.

In some embodiments, the gene product that disrupts a translesion synthesis pathway is an umuD′, umuC, recA, dinB, or polB gene product. In some embodiments, the recA gene product is a recA730 gene product. In some embodiments, the polB gene product is a polB(D156A) gene product. In some embodiments, the gene product that disrupts a methyl-directed mismatch repair pathway is a mutS, mutL, mutH, dam, or seqA gene product. In some embodiments, the mutS gene product is a mutS538, mutS503, or mutSΔN gene product. In some embodiments, the mutL gene product is a mutL705, mutL713, mutL(R261H), or mutL(K307A) gene product. In some embodiments, the mutH gene product is a mutH(E56A), mutH(K79E), or mutH(K 116E) gene product. In some embodiments, the gene product that disrupts a base excision repair pathway is a ugi, AID, APOBEC, CDA, MAG, or AAG gene product. In some embodiments, the AID gene product is an AID (7), AID (7.3), AID (7.3.5), AID (7.3.3), AID (7.3.1), or AID (7.3.2) gene product. In some embodiments, the APOBEC gene product is an APOBEC 1 gene product. In some embodiments, the CDA gene product is a CDA1 gene product. In some embodiments, the MAG gene product is a MAG1 gene product. In some embodiments, the AAG gene product is an AAG(Y127I-H136L) or Δ80-AAG(Y127I-H136L) gene product.

In some embodiments, the gene product that disrupts a base selection pathway is a dnaE74, dnaE486, dnaE1026, dnaX36, dnaX2016, emrR, nrdAB, nrdA(H59A)B, nrdA(A65V)B, nrdA(A301V)B, nrdAB(P334L), or nrdEF gene product.

In some embodiments, the expression construct comprises a nucleic acid sequence encoding a rsmE, cchA, yffI, or yfjY gene product.

In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaQ926 gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a umuD′, umuC, or recA730 gene product, or any combination thereof. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a dnaE486, dnaE1026, dnaX36, or dnaX2016 gene product, or any combination thereof. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a mutS538, mutS503, mutL705, mutL713, mutL(R261H), mutL4K307A), mutH(E56A), mutH(K79E), or mutH(K116E) gene product, or any combination thereof. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a Dam gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a seqA gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a emrR, mutH(E56A), mutL713, mutS503, mutSΔN, dinB, or polB gene product, or any combination thereof.

In some embodiments, the expression construct comprises a nucleic acid sequence encoding a Dam gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a seqA gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a emrR gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a ugi gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding an AID, AID (7), AID (7.3), AID (7.3.5). AID (7.3.3), AID (7.3.1), AID (7.3.2), APOBEC1, or CDA1 gene product, or any combination thereof. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a mutSΔN gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a rsmE, cchA, yffI, yfjY, nrdAB, nrdA(H59A)B, nrdA(A65V)B, nrdA(A30IV)B, nrdEF, or nrdAB(P334L) gene product, or any combination thereof.

In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaE74 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaE486 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaE1026 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaX36 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaX2016 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a rpsD12 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a rpsD14 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a rpsD16 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a polB gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a polB(D156A) gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a MAG1 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a AAG(Y127I-H136L) gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a Δ80-AAG(Y127I-H136L) gene product.

In some embodiments, the expression construct is comprised in a plasmid. In some embodiments, the plasmid comprises a bacterial origin of replication. In some embodiments, the origin of replication is a cloDF13 origin of replication. In some embodiments, the plasmid comprises a nucleic acid sequence encoding a gene product conferring resistance to an antibiotic to a bacterial host cell. In some embodiments, the antibiotic is chloramphenicol, kanamycin, tetracycline, tetracycline, spectinomycin or apramycin, or ampicillin. Additional suitable origins of replication and antibiotics for use in embodiments of this disclosure will be apparent to those of skill in the art. The disclosure is not limited in this respect.

In some embodiments, the expression construct comprises at least one inducible promoter controlling the expression of at least one nucleic acid sequence encoding the gene product that disrupts a proofreading pathway, a translesion synthesis pathway, a methyl-directed mismatch repair pathway, a base excision repair pathway, or a base selection pathway, or any combination thereof. In some embodiments, the inducible prompter is an arabinose responsive promoter. In some embodiments, the arabinose responsive promoter is a PBAD promoter. In some embodiments, the expression construct comprises a nucleic acid sequence encoding an arabinose operon regulatory protein. In some embodiments, the arabinose operon regulatory protein is araC. In some embodiments, the nucleic acid sequence encoding the arabinose operon regulatory protein is under the control of a weak promoter. In some embodiments, the weak promoter is a PC promoter. Additional weak promoters will be apparent to those of skill in the art, and the present disclosure is not limited in this respect.

In some embodiments, the expression construct comprises at least one codon-optimized nucleic acid sequence encoding a gene product. In some embodiments, the codon-optimized nucleic acid sequence comprises at least one codon of a naturally-occurring sequence encoding the gene product that has been replaced with a different codon encoding the same amino acid. In some embodiments, the at least one codon replacing the codon of the naturally occurring nucleic acid sequence corresponds to a tRNA expressed in a bacterial host cell. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a ribosome-binding site, and wherein at least one ribosome-binding site encoded by the expression construct is modified as compared to a naturally occurring ribosome binding site. In some embodiments, the modified ribosome binding site exhibits increased ribosome binding as compared to the naturally occurring ribosome binding site.

In some embodiments, the expression construct comprises a nucleic acid sequence encoding a gene product or a combination of such sequences listed in Table 2 or FIG. 7.

Some aspects of this disclosure provide plasmids, cosmids, or artificial chromosomes (e.g., bacterial artificial chromosome, yeast artificial chromosomes, etc.) comprising an expression construct as disclosed herein.

Some aspects of this disclosure provide a cell comprising an expression construct or a plasmid as provided herein. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell further comprises a selection plasmid or an accessory plasmid. In some embodiments, the cell is a host cell for a bacteriophage. In some embodiments, the cell is an E. coli cell. In some embodiments, the cell is in a lagoon.

Some aspects of this disclosure provide methods for modulating the mutation rate in a cell, e.g., in a host cell for a bacteriophage. In some embodiments, the method comprises contacting the cell with an expression construct or a plasmid as disclosed herein. In some embodiments, the expression construct comprises an inducible promoter, and the method further comprises culturing the host cell under conditions suitable to induce expression from the inducible promoter. In some embodiments, the inducible promoter is an arabinose-inducible promoter, and culturing the host cell under conditions suitable to induce expression from the inducible promoter comprises contacting the host cell with an amount of arabinose sufficient to increase expression of the arabinose-inducible promoter by at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, at least 10000-fold, at least 50000-fold, at least 100000-fold, at least 500000-fold, or at least 1000000-fold as compared to basal expression in the absence of arabinose. In some embodiments, the method results in an at least 10-fold, at least 100-fold, at least 1000-fold, at least 10000-fold, at least 15000-fold, at least 200000-fold, at least 250000-fold, or at least 300000-fold increased mutation rate as compared to the mutation rate in the host cell in the absence of the expression construct.

Some aspects of this disclosure provide methods for directed evolution using an expression construct provided herein. In some embodiments, the method comprises (a) contacting a population of host cells comprising a mutagenesis expression construct or plasmid as provided herein with a population of phage vectors comprising a gene to be evolved and deficient in at least one gene for the generation of infectious phage particles, wherein (1) the host cells are amenable to transfer of the vector; (2) the vector allows for expression of the gene to be evolved in the host cell, can be replicated by the host cell, and the replicated vector can transfer into a second host cell; (3) the host cell expresses a gene product encoded by the at least one gene for the generation of infectious phage particles of (a) in response to the activity of the gene to be evolved, and the level of gene product expression depends on the activity of the gene to be evolved; (b) incubating the population of host cells under conditions allowing for mutation of the gene to be evolved and the transfer of the vector comprising the gene to be evolved from host cell to host cell, wherein host cells are removed from the host cell population, and the population of host cells is replenished with fresh host cells that comprise the expression construct but do not harbor the vector, and (c) isolating a replicated vector from the host cell population in (b), wherein the replicated vector comprises a mutated version of the gene to be evolved.

In some embodiments, the expression construct comprises an inducible promoter, and the step of incubating (step (b)) comprises culturing the population of host cells under conditions suitable to induce expression from the inducible promoter. In some embodiments, the inducible promoter is an arabinose-inducible promoter, and the step of incubating (step (b)) comprises contacting the host cell with an amount of arabinose sufficient to increase expression of the arabinose-inducible promoter by at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, at least 10000-fold, at least 50000-fold, at least 100000-fold, at least 500000-fold, or at least 1000000-fold as compared to basal expression in the absence of arabinose.

In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a phage. In some embodiments, the phage is a filamentous phage. In some embodiments, the phage is an M13 phage.

In some embodiments, the at least one gene for the generation of infectious phage particles comprises a sequence encoding a pIII protein. In some embodiments, the at least one gene for the generation of infectious phage particles comprises a full-length gIII gene. In some embodiments, the host cells comprise all phage genes except for the at least one gene for the generation of infectious phage particles in the form of a helper phage. In some embodiments, the phage genes comprised on the helper phage comprise pI, pII, pIV, pV, pVI, pVII, pVIII, pIX, pX, and/or pXI.

In some embodiments, the host cells comprise an accessory plasmid. In some embodiments, the accessory plasmid comprises an expression construct encoding the pIII protein under the control of a promoter that is activated by a gene product encoded by the gene to be evolved. In some embodiments, the host cells comprise the accessory plasmid, and together the helper phage and the accessory plasmid comprise all genes required for the generation of an infectious phage. In some embodiments, the method further comprises a negative selection for undesired activity of the gene to be evolved. In some embodiments, the host cells comprise an expression construct encoding a dominant-negative pII protein (pIII-neg). In some embodiments, expression of the pIII-neg protein is driven by a promoter the activity of which depends on an undesired function of the gene to be evolved.

In some embodiments, step (b) comprises incubating the population of host cells for a time sufficient for at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least, 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 7500, at least 10000, or more consecutive life cycles of the viral vector or phage. In some embodiments, the host cells are E. coli cells.

In some embodiments, the host cells are incubated in suspension culture. In some embodiments, the population of host cells is continuously replenished with fresh host cells that do not comprise the vector. In some embodiments, fresh cells are being replenished and cells are being removed from the cell population at a rate resulting in a substantially constant number of cells in the cell population. In some embodiments, fresh cells are being replenished and cells are being removed from the cell population at a rate resulting in a substantially constant vector population. In some embodiments, fresh cells are being replenished and cells are being removed from the cell population at a rate resulting in a substantially constant vector, viral, or phage load. In some embodiments, the rate of fresh cell replenishment and/or the rate of cell removal is adjusted based on quantifying the cells in the cell population. In some embodiments, the rate of fresh cell replenishment and/or the rate of cell removal is adjusted based on quantifying the frequency of host cells harboring the vector and/or of host cells not harboring the vector in the cell population. In some embodiments, the quantifying is by measuring the turbidity of the host cell culture, measuring the host cell density, measuring the wet weight of host cells per culture volume, or by measuring light extinction of the host cell culture.

In some embodiments, the host cells are not exposed to a mutagen other than the mutagenesis expression construct or constructs provided herein. In some embodiments, the host cells are exposed to a mutagen. In some embodiments, the mutagen is ionizing radiation, ultraviolet radiation, base analogs, deaminating agents (e.g., nitrous acid), intercalating agents (e.g., ethidium bromide), alkylating agents (e.g., ethylnitrosourea), transposons, bromine, azide salts, psoralen, benzene,3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) (CAS no. 77439-76-0), O,O-dimethyl-S-(phthalimidomethyl)phosphorodithioate (phos-met) (CAS no. 732-11-6), formaldehyde (CAS no. 50-00-0), 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) (CAS no. 3688-53-7), glyoxal (CAS no. 107-22-2), 6-mercaptopurine (CAS no. 50-44-2), N-(trichloromethylthio)-4-cyclohexane-1,2-dicarboximide (captan) (CAS no. 133-06-2), 2-aminopurine (CAS no. 452-06-2), methyl methane sulfonate (MMS) (CAS No. 66-27-3), 4-nitroquinoline 1-oxide (4-NQO) (CAS No. 56-57-5), N4-Aminocytidine (CAS no. 57294-74-3), sodium azide (CAS no. 26628-22-8), N-ethyl-N-nitrosourea (ENU) (CAS no. 759-73-9), N-methyl-N-nitrosourea (MNU) (CAS no. 820-60-0), 5-azacytidine (CAS no. 320-67-2), cumene hydroperoxide (CHP) (CAS no. 80-15-9), ethyl methanesulfonate (EMS) (CAS no. 62-50-0), N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) (CAS no. 4245-77-6), N-methyl-N-nitro-N-nitrosoguanidine (MNNG) (CAS no. 70-25-7), 5-diazouracil (CAS no. 2435-76-9), or t-butyl hydroperoxide (BHP) (CAS no. 75-91-2).

In some embodiments, the method comprises a phase of diversifying the population of vectors by mutagenesis, in which the cells are incubated under conditions suitable for mutagenesis of the gene to be evolved in the absence of stringent selection for vectors having acquired a gain-of-function mutation in the gene to be evolved. In some embodiments, the method comprises a phase of stringent selection for a mutated replication product of the viral vector encoding an evolved gene.

Some aspects of this disclosure provide kits comprising (a) a mutagenesis expression construct or plasmid as provided herein, wherein the expression construct comprises an inducible promoter controlling at least one of the nucleic acid sequences comprised in the expression construct; and (b) an inducing agent that induces expression from the inducible promoter. In some embodiments, the kit further comprises (c) a vector encoding an M13 phage backbone and a multiple cloning site for insertion of a nucleic acid sequence encoding a gene product to be evolved, wherein the vector or a replication product thereof can be packaged into infectious phage particles in the presence of other phage functions by suitable host cells, but lacks at least one gene required for the generation of infectious particles. In some embodiments, the kit further comprises (d) an accessory plasmid comprising a nucleic acid sequence encoding the at least one gene required for the generation of infectious particles under the control of a promoter that is activated by a desired activity of the gene product to be evolved. In some embodiments, the kit further comprises (e) an accessory plasmid comprising a nucleic acid sequence encoding a dominant-negative version of the at least one gene required for the generation of infectious particles under the control of a promoter that is activated by an undesired activity of the gene product to be evolved. In some embodiments, the kit further comprises a helper phage providing all phage functions except for the at least one gene required for the generation of infectious phage particles provided by the accessory plasmid of (d). In some embodiments, the helper phage or a replication product thereof cannot be packaged into infectious phage particles. In some embodiments, the kit comprises suitable host cells. In some embodiments, the host cells are E. coli host cells.

The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, the Drawings, the Examples, and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-IB. Mutagenesis plasmid (MP) design and effect on mutation rate in bacteria. The mutator genes tested are color-coded to indicate the canonical pathway being disrupted through the overexpression (FIG. 1A) or deletion (FIG. 1B) of that gene. All MPs express mutator genes from the arabinose-inducible PBAD promoter, with each gene preceded by a ribosome-binding site to allow for translation from a single transcript. For (FIG. 1A), the mutagenesis rate μbp (substitutions per base pair of the E. coli genome per generation) was calculated using rifampin resistance under uninduced (25 mM glucose, white bars) and induced (25 mM glucose+25 mM arabinose, black bars). For (FIG. 1B), the rifampin resistance of XL1-Blue (white bar) and XL1-Red (black bar) was used to calculate μbp.

FIGS. 2A-2C. Features of the MP system. (FIG. 2A) The dynamic range of MP6 was evaluated using increasing concentrations of arabinose (black circles) in the presence of 25 mM glucose in all cases. Treatment with 200 mM glucose only (dotted line) showed low mutagenesis under identical conditions. Comparison of the mutation rate under induced and uninduced conditions reveals a 35,000-fold dynamic range. Using the number of unique mutations found in rifampin-resistant rpoB alleles (21 sites), the average number of substitutions/genome/generation (μg) was calculated for (FIG. 2B) MG1655 ΔrecA::apra without an MP (white bar) vs. carrying MP1-6 under induced conditions (black bars), and (FIG. 2C) XL1-Blue (white bar) vs. XL1-Red (black bar).

FIGS. 3A-3C. In vivo mutagenesis of M13 bacteriophage DNA. (FIG. 3A) S1030 cells (F+) carrying the indicated MPs were infected with M13 bacteriophage carrying a constitutive lacZ expression cassette (SP063), accompanied by the induction of the MP using arabinose, or suppression of the MP using glucose. (FIG. 3B) XL1-Blue or XL1-Red cells were transformed with purified SP063 DNA and recovered after overnight growth. (FIG. 3C) S1021 cells (identical to S1030, but lacking F′) carrying the indicated MPs were induced with arabinose or repressed with glucose for 2 h, transformed with purified SP063 DNA, and again induced with arabinose or repressed with glucose during recovery. For (FIG. 3A)-(FIG. 3C), progeny phage from the overnight propagation were plaqued on S1030 cells and stained using the X-Gal analog Bluo-Gal. The fraction of plaques that showed a white or light-blue lacZ− phenotype reflecting loss-of-function mutation(s) is shown.

FIGS. 4A-4I. Mutagenic spectra of the MPs. (FIGS. 4A-4D) The rpoB locus of single rifampin-resistant colonies was amplified by PCR and sequenced in both clusters I (aa 451-754) and II (aa 84-401). (FIG. 4E) Identities of the rifampin-resistant rpoB alleles from MP1, MP4, or MP6 mutagenesis, or using XL1-Red. The MPs yielded a wide distribution of mutation types, with the diversity of alleles strongly correlating with MP potency. (FIGS. 4F-4I) SP063 phage containing a constitutive lacZ expression cassette was propagated on the indicated mutator strain under induced conditions, and mutations were analyzed by high-throughput sequencing. In all cases, the number of sequenced mutations (n) is indicated for the MP and XL1-Red assays. * For (FIG. 4D) and (FIG. 4E), all sequenced XL1-Red rifampin-resistant colonies carried the identical F505S/S531F rpoB genotype.

FIGS. 5A-5B. Comparison of MP6 and other mutagenesis approaches for the evolution of antibiotic resistance. (FIG. 5A) MG1655 ΔrecA::apra cells with or without MP6 were grown for 18-21 h under induced conditions. XL1-Blue and XL1-Red were grown for 18-21 h. (FIG. 5B) MG1655 ΔrecA::apra cells were treated using chemical mutagens as previously described 1321. In all cases, cultures were plated on the indicated antibiotics following overnight culture in the absence of any selection. The numbers in parenthesis indicate the antibiotic concentrations used in μg/mL. The fraction of cells resistant to each antibiotic was calculated relative to the total number of cells on plates without antibiotics. Resistance to norfloxacin was not detected for any of the tested strains or treatments. XL1-Blue and XL1-Red are both inherently resistant to tetracycline and metronidazole, so no comparison is shown for either antibiotic. See Table 6 for full antibiotic resistance data.

FIGS. 6A-6B. Continuous evolution of PT3-active RNAP variants. S1030 cells carrying an accessory plasmid (AP) encoding PT3 upstream of M13 bacteriophage geneIII and either MP1a or MP6a were infected with selection phage (SP) carrying wild-type T7 RNAP under conditions in which selection stringency was high (0 ng/mL ATc) or moderate (30 ng/mL ATc). (FIG. 6A) Total phage titers at 10 h and 20 h after lagoon inoculation with the SP encoding T7 RNAP. (FIG. 6B) Titers of phage encoding evolved RNAP variants active on PT3. The limit of detection is 50 pfu/mL.

FIG. 7. Summary of major pathways that influence E. coli DNA replication fidelity. Steps during replication and mutation correction are grouped according to their mechanisms of action. Methylated DNA is shown in black, unmethylated DNA is shown in grey, and the mutation to be corrected is depicted as “M”. Gene superscripts denote if a mutator phenotype results upon gene deletion [43] (1), gene overexpression [17, 20, 25, 46, 48] (2) or modification of the chromosomal allele to circumvent potential knockout lethality [17, 20, 25, 46, 48, 49] (3).

FIG. 8. Cryptic σ70 promoter at the 3′ end of the dnaQ926 ORF. Annotated sequence of the predicted σ70 promoter in MP3 bridging the 3′ end of the dnaQ926 ORF and RBS driving the dam ORF. Sequences correspond, from top to bottom, to SEQ ID NOs: 12-15.

FIG. 9. Cryptic σ70 promoters at the 3′ end of the seqA ORF. Annotated sequence of the predicted σ70 promoters in MP5 bridging the 3′ end of the seqA ORF and native RBS driving the ugi ORF. Sequences correspond, from top to bottom, to SEQ ID NOs: 16-19.

FIGS. 10A-10B. Effect of MPs on host viability under induced conditions. (FIG. 10A) Relative cell viability was calculated as the fractional cell titer following arabinose induction as compared to the uninduced control for each MP. Viability is anti-correlated with mutagenic potency at high levels. (FIG. 10B) The XL1-Red strain shows the expected level of viability (compared to the control, XL1-Blue) given its mutagenic potency as compared to the designed MPs.

FIG. 11. Relationship between host viability and induced levels of mutagenesis for all MPs. Low potency MPs which induce up to ˜1×10−6, substitutions/bp/generation were well tolerated by the E. coli MG1655 ΔrecA::apra host, while higher levels of mutagenesis generally resulted in a reduced host viability, as expected. This inflection point corresponds to ˜4.6 substitutions/genome/generation for wild-type E. coli MG/566 (genome size=4.64×106 bp).

FIG. 12. Relationship between uninduced and induced levels of mutagenesis for all MPs. Higher levels of background (uninduced mutagenesis) were generally accompanied by an increase in overall MP mutagenesis upon induction. The full data set is provided in Table 2.

FIG. 13. Plaque assay of the lacZ-carrying M13 phage SP063. SP063 carries the wild-type E. coli β-galactosidase gene with a consensus ribosome-binding site directly downstream of geneIII. Plating using soft agar containing S1030 cells in the presence of the X-Gal analog Bluo-Gal (Life Technologies) results in a strong, deep blue plaques (shown here as dark circles).

FIGS. 14A-14B. Optimization of phage inoculant for optimal expansion and mutagenesis. (FIG. 14A) E. coli S1030 carrying MP6 were induced with arabinose during log-phase growth and concomitantly infected with serially diluted SP063 phage. Phage were titered after overnight propagation and the fold expansion of the phage population was determined. (FIG. 14B) The percentage of white and light blue (lacZ−) plaques in the presence of Bluo-Gal suggests a correlation between phage population expansion and mutagenesis efficiency.

FIGS. 15A-15B. Effect of MP pre-induction on phage mutagenesis. (FIG. 15A) E. coli S1030 carrying MP6 were induced with arabinose during log-phase growth and concomitantly infected with SP063 phage at defined titers. Alternatively, infection was delayed for 1 h or 2 h. (FIG. 15B) Phage were titered after overnight propagation and the percentage of white plaques in the presence of Bluo-Gal was counted.

FIG. 16. Analysis of F′ episomal mutations rates using various MPs. The frequency of lacZ+ revertants is the fraction of colonies surviving on lactose as the sole carbon source as compared to the total colony count (colonies that survive on glucose as sole carbon source). Each strain reports the MP's ability to increase the frequency of a specific mutation type.

FIGS. 17A-17D. Mutagenic spectra of commonly used mutagenesis techniques. Previously reported mutagenic spectra of four commonly used mutagenesis methods are shown: FIG. 17A: LF Pol I9; FIG. 17B: mutA37; FIG. 17C: Mutazyme II38; FIG. 17D: and EMS39.

FIG. 18. Activity of T7 RNAP on cognate and non-cognate promoters. Log-phase S1030 cells carrying accessory plasmids (APs) with a geneIII cassette with an upstream phage shock protein (PSP). T7, hybrid T7/T3, or T3 promoter were infected with selection phage (SPs) carrying the wild-type T7 RNAP. The fraction of output phage vs. input phage indirectly reports on the activity of the T7 RNAP on the various promoters. Enrichment factors of ˜100 or less indicate extremely weak to non-existent activity.

FIG. 19. Single-phage plaque sequencing of PT3-active SPs. Single phage plaques at 10 h of PACE using the PT3 AP were isolated and subjected to Sanger sequencing. All clones carried T7 RNAP variants with conserved mutations known to confer activity on PT3 (blue), as well as additional mutations that may further enhance activity (black). Silent mutations were also detected (red).

FIG. 20. DP1 Vector Map. A schematic depiction of one embodiment of a DP1 drift plasmid is provided, referenced herein as SEQ ID NO: 27. This embodiment comprises araC, dnaQ926, umuD′, umuC, recA730, and an anhydrotetracycline (ATc)-dependent drift promoter.

FIG. 21. DP2 Vector Map. A schematic depiction of one embodiment of a DP2 drift vector is provided, referenced herein as SEQ ID NO: 28. This embodiment comprises araC, dnaQ926, and an anhydrotetracycline (ATc)-dependent drift promoter.

FIG. 22. DP3 Vector Map. A schematic depiction of one embodiment of a DP3 drift vector is provided, referenced herein as SEQ ID NO: 29. This embodiment comprises araC, dnaQ926, dam, and an anhydrotetracycline (ATc)-dependent drift promoter.

FIG. 23. DP4 Vector Map. A schematic depiction of one embodiment of a DP4 drift vector is provided, referenced herein as SEQ ID NO: 33. This embodiment comprises araC, dnaQ926, dam, seqA, and an anhydrotetracycline (ATc)-dependent drift promoter.

FIG. 24. DP5 Vector Map. A schematic depiction of one embodiment of a DP5 drift vector is provided, referenced herein as SEQ ID NO: 34. This embodiment comprises araC, dnaQ926, dam, seqA, ugi and/or pmCDA1, and an anhydrotetracycline (ATc)-dependent drift promoter.

FIG. 25. DP6 Vector Map. A schematic depiction of one embodiment of a DP6 drift vector is provided, referenced herein as SEQ ID NO: 35. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and/or pmCDA1, and an anhydrotetracycline (ATc)-dependent drift promoter.

FIG. 26. MP1 Vector Map. A schematic depiction of one embodiment of a MP1 mutagenesis vector is provided, referenced herein as SEQ ID NO: 43. This embodiment comprises dnaQ926, umuD′, umuC, and recA730.

FIG. 27. MP2 Vector Map. A schematic depiction of one embodiment of a MP2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 44. This embodiment comprises dnaQ926.

FIG. 28. MP3 Vector Map. A schematic depiction of one embodiment of a MP3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 45. This embodiment comprises araC, dnaQ926, and dam.

FIG. 29. MP4 Vector Map. A schematic depiction of one embodiment of a MP4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 46. This embodiment comprises araC, dnaQ926, dam, and seqA.

FIG. 30. MP5 Vector Map. A schematic depiction of one embodiment of a MP5 mutagenesis vector is provided, referenced herein as SEQ ID NO: 47. This embodiment comprises araC, dnaQ926, dam, seqA, ugi and pmCDA1

FIG. 31. MP6 Vector Map. A schematic depiction of one embodiment of a MP6 mutagenesis vector is provided, referenced herein as SEQ ID NO: 48. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and pmCDA1.

FIG. 32. MP-B2 Vector Map. A schematic depiction of one embodiment of a MP-B2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 49. This embodiment comprises dnaE374.

FIG. 33. MP-B4 Vector Map. A schematic depiction of one embodiment of a MP-B4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 50. This embodiment comprises dnaE486.

FIG. 34. MP-B5 Vector Map. A schematic depiction of one embodiment of a MP-B5 mutagenesis vector is provided, referenced herein as SEQ ID NO: 51. This embodiment comprises araC, and dnaE1026.

FIG. 35. MP-C2 Vector Map. A schematic depiction of one embodiment of a MP-C2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 52. This embodiment comprises araC, and dnaX36.

FIG. 36. MP-C3 Vector Map. A schematic depiction of one embodiment of a MP-C3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 53. This embodiment comprises araC, and dnaX2016.

FIG. 37. MP-D3 Vector Map. A schematic depiction of one embodiment of a MP-D3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 54. This embodiment comprises araC, and dnaE486.

FIG. 38. MP-D4 Vector Map. A schematic depiction of one embodiment of a MP-D4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 55. This embodiment comprises araC, dnaQ926, and dnaE1026.

FIG. 39. MP-E Vector Map. A schematic depiction of one embodiment of a MP-E mutagenesis vector is provided, referenced herein as SEQ ID NO: 56. This embodiment comprises araC, dnaQ926, and dnaX36.

FIG. 40. MP-E2 Vector Map. A schematic depiction of one embodiment of a MP-E2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 57. This embodiment comprises araC, dnaQ926, and dnaX2016.

FIG. 41. MP-F2 Vector Map. A schematic depiction of one embodiment of a MP-F2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 58. This embodiment comprises araC, dnaQ926, dnaX2016, and mutS538′.

FIG. 42. MP-F3 Vector Map. A schematic depiction of one embodiment of a MP-F3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 59. This embodiment comprises araC, dnaQ926, and mutS503′.

FIG. 43. MP-H Vector Map. A schematic depiction of one embodiment of a MP-H mutagenesis vector is provided, referenced herein as SEQ ID NO: 60. This embodiment comprises araC, dnaQ926, and mutL705.

FIG. 44. MP-H2 Vector Map. A schematic depiction of one embodiment of a MP-H2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 61. This embodiment comprises araC, dnaQ926, and mutL713.

FIG. 45. MP-H3 Vector Map. A schematic depiction of one embodiment of a MP-H3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 62. This embodiment comprises araC, dnaQ926, and mutL(R261H).

FIG. 46. MP-H4 Vector Map. A schematic depiction of one embodiment of a MP-H4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 63. This embodiment comprises araC, dnaQ926, and mutL(K307A).

FIG. 47. MP-I Vector Map. A schematic depiction of one embodiment of a MP-I mutagenesis vector is provided, referenced herein as SEQ ID NO: 64. This embodiment comprises araC, dnaQ926, and mutH (E564).

FIG. 48. MP-I2 Vector Map. A schematic depiction of one embodiment of a MP-I2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 65. This embodiment comprises araC, dnaQ926, and mutH (K79E).

FIG. 49. MP-I3 Vector Map. A schematic depiction of one embodiment of a MP-I3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 66. This embodiment comprises araC, dnaQ926, and mutH (K116E).

FIG. 50. MP-J Vector Map. A schematic depiction of one embodiment of a MP-J mutagenesis vector is provided, referenced herein as SEQ ID NO: 67. This embodiment comprises araC, and rpsD12.

FIG. 51. MP-J2 Vector Map. A schematic depiction of one embodiment of a MP-J2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 68. This embodiment comprises araC, and rpsD14.

FIG. 52. MP-J3 Vector Map. A schematic depiction of one embodiment of a MP-J3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 69. This embodiment comprises araC, and rpsD16.

FIG. 53. MP-K10 Vector Map. A schematic depiction of one embodiment of a MP-K10 mutagenesis vector is provided, referenced herein as SEQ ID NO: 70. This embodiment comprises araC, dnaQ926, dam, seqA, and emrR.

FIG. 54. MP-K11 Vector Map. A schematic depiction of one embodiment of a MP-K11 mutagenesis vector is provided, referenced herein as SEQ ID NO: 71. This embodiment comprises araC, dnaQ926, dam, seqA, and mutSdeIN (del 2-98).

FIG. 55. MP-K12 Vector Map. A schematic depiction of one embodiment of a MP-K12 mutagenesis vector is provided, referenced herein as SEQ ID NO: 72. This embodiment comprises araC, dnaQ926, dam, seqA, and dinB.

FIG. 56. MP-K13 Vector Map. A schematic depiction of one embodiment of a MP-K13 mutagenesis vector is provided, referenced herein as SEQ ID NO: 73. This embodiment comprises araC, dnaQ926, dam, seqA, and polB.

FIG. 57. MP-K14 Vector Map. A schematic depiction of one embodiment of a MP-K14 mutagenesis vector is provided, referenced herein as SEQ ID NO: 74. This embodiment comprises araC, dnaQ926, dam, and seqA (RBS+).

FIG. 58. MP-K7 Vector Map. A schematic depiction of one embodiment of a MP-K7 mutagenesis vector is provided, referenced herein as SEQ ID NO: 75. This embodiment comprises araC, dnaQ926, dam, and emrR.

FIG. 59. MP-K9 Vector Map. A schematic depiction of one embodiment of a MP-K9 mutagenesis vector is provided, referenced herein as SEQ ID NO: 76. This embodiment comprises araC, dnaQ926, dam, and mutSdeIN(2-98).

FIG. 60. MP-L Vector Map. A schematic depiction of one embodiment of a MP-L mutagenesis vector is provided, referenced herein as SEQ ID NO: 77. This embodiment comprises araC, and polB.

FIG. 61. MP-L2 Vector Map. A schematic depiction of one embodiment of a MP-L2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 78. This embodiment comprises araC, and polB(D156A).

FIG. 62. MP-P Vector Map. A schematic depiction of one embodiment of a MP-P mutagenesis vector is provided, referenced herein as SEQ ID NO: 79. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, and mutH(E56A).

FIG. 63. MP-P11 Vector Map. A schematic depiction of one embodiment of a MP-P11 mutagenesis vector is provided, referenced herein as SEQ ID NO: 80. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and pmCDA1, mutSdeIN(del-29).

FIG. 64. MP-P3 Vector Map. A schematic depiction of one embodiment of a MP-P3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 81. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, and mutL713.

FIG. 65. MP-P4 Vector Map. A schematic depiction of one embodiment of a MP-P4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 82. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, and mutS503′.

FIG. 66. MP-P5 Vector Map. A schematic depiction of one embodiment of a MP-P5 mutagenesis vector is provided, referenced herein as SEQ ID NO: 83. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, and mutSdeIN(del 2-98).

FIG. 67. MP-P6 Vector Map. A schematic depiction of one embodiment of a MP-P6 mutagenesis vector is provided, referenced herein as SEQ ID NO: 84. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, and dinB.

FIG. 68. MP-P7 Vector Map. A schematic depiction of one embodiment of a MP-P7 mutagenesis vector is provided, referenced herein as SEQ ID NO: 85. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, and polB.

FIG. 69. MP-P8 Vector Map. A schematic depiction of one embodiment of a MP-P8 mutagenesis vector is provided, referenced herein as SEQ ID NO: 86. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and hsAID.

FIG. 70. MP-P9 Vector Map. A schematic depiction of one embodiment of a MP-P9 mutagenesis vector is provided, referenced herein as SEQ ID NO: 87. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and mAPOBEC1.

FIG. 71. MP-Q Vector Map. A schematic depiction of one embodiment of a MP-Q mutagenesis vector is provided, referenced herein as SEQ ID NO: 88. This embodiment comprises araC, dnaQ926, dam, seqA, and rsmE.

FIG. 72. MP-Q10 Vector Map. A schematic depiction of one embodiment of a MP-Q10 mutagenesis vector is provided, referenced herein as SEQ ID NO: 89. This embodiment comprises araC, dnaQ926, dam, seqA, and nrdA(A65V)B.

FIG. 73. MP-Q11 Vector Map. A schematic depiction of one embodiment of a MP-Q11 mutagenesis vector is provided, referenced herein as SEQ ID NO: 90. This embodiment comprises araC, dnaQ926, dam, seqA, and nrdA(A301V)B.

FIG. 74. MP-Q12 Vector Map. A schematic depiction of one embodiment of a MP-Q12 mutagenesis vector is provided, referenced herein as SEQ ID NO: 91. This embodiment comprises araC, dnaQ926, dam, seqA, and nrdAB(P334L).

FIG. 75. MP-Q13 Vector Map. A schematic depiction of one embodiment of a MP-Q13 mutagenesis vector is provided, referenced herein as SEQ ID NO: 92. This embodiment comprises araC, dnaQ926, dam, seqA, and nrdEF.

FIG. 76. MP-Q2 Vector Map. A schematic depiction of one embodiment of a MP-Q2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 93. This embodiment comprises araC, dnaQ926, dam, seqA, and cchA.

FIG. 77. MP-Q3 Vector Map. A schematic depiction of one embodiment of a MP-Q3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 94. This embodiment comprises araC, dnaQ926, dam, seqA, and yffI.

FIG. 78. MP-Q4 Vector Map. A schematic depiction of one embodiment of a MP-Q4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 95. This embodiment comprises araC, dnaQ926, dam, seqA, and yfjY.

FIG. 79. MP-Q5 Vector Map. A schematic depiction of one embodiment of a MP-Q5 mutagenesis vector is provided, referenced herein as SEQ ID NO: 96. This embodiment comprises araC, dnaQ926, dam, seqA, ugi and hsAID.

FIG. 80. MP-Q6 Vector Map. A schematic depiction of one embodiment of a MP-Q6 mutagenesis vector is provided, referenced herein as SEQ ID NO: 97. This embodiment comprises araC, dnaQ926, dam, seqA, ugi and mAPOBEC1.

FIG. 81. MP-Q8 Vector Map. A schematic depiction of one embodiment of a MP-Q8 mutagenesis vector is provided, referenced herein as SEQ ID NO: 98. This embodiment comprises araC, dnaQ926, dam, seqA, and nrdAB.

FIG. 82. MP-Q9 Vector Map. A schematic depiction of one embodiment of a MP-Q9 mutagenesis vector is provided, referenced herein as SEQ ID NO: 99. This embodiment comprises araC, dnaQ926, dam, seqA, and nrdA(H59A)B.

FIG. 83. MP-R Vector Map. A schematic depiction of one embodiment of a MP-R mutagenesis vector is provided, referenced herein as SEQ ID NO: 100. This embodiment comprises araC, dnaQ926, dam, seqA, ugi and hsAID.

FIG. 84. MP-R2 Vector Map. A schematic depiction of one embodiment of a MP-R2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 101. This embodiment comprises araC, dnaQ926, dam, seqA, ugi and mAPOBEC1.

FIG. 85. MP-R3 Vector Map. A schematic depiction of one embodiment of a MP-R3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 102. This embodiment comprises araC, dnaQ926, dam, seqA, ugi and pmCDA1.

FIG. 86. MP-R4 Vector Map. A schematic depiction of one embodiment of a MP-R4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 103. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and hsAID.

FIG. 87. MP-R5 Vector Map. A schematic depiction of one embodiment of a MP-R5 mutagenesis vector is provided, referenced herein as SEQ ID NO: 104. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and mAPOBEC1.

FIG. 88. MP-R6 Vector Map. A schematic depiction of one embodiment of a MP-R6 mutagenesis vector is provided, referenced herein as SEQ ID NO: 105. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and pmCDA1.

FIG. 89. MP-S Vector Map. A schematic depiction of one embodiment of a MP-S mutagenesis vector is provided, referenced herein as SEQ ID NO: 106. This embodiment comprises araC, dnaQ926, and MAG1.

FIG. 90. MP-S2 Vector Map. A schematic depiction of one embodiment of a MP-S2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 107. This embodiment comprises araC, dnaQ926, and AAG(Y127I-H136L).

FIG. 91. MP-S3 Vector Map. A schematic depiction of one embodiment of a MP-S3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 108. This embodiment comprises araC, dnaQ926, and del80-AAG(Y127I-H136L).

FIG. 92. MP-T Vector Map. A schematic depiction of one embodiment of a MP-T mutagenesis vector is provided, referenced herein as SEQ ID NO: 109. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and hsAID (K10E/E156G).

FIG. 93. MP-T2 Vector Map. A schematic depiction of one embodiment of a MP-T2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 110. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and hsAID (K10E/E156G/T821).

FIG. 94. MP-T3 Vector Map. A schematic depiction of one embodiment of a MP-T3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 111. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and hsAID (K10E/E156G/T821).

FIG. 95. MP-T4 Vector Map. A schematic depiction of one embodiment of a MP-T4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 112. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and hsAID (K10E/E156G/T821).

FIG. 96. MP-T5 Vector Map. A schematic depiction of one embodiment of a MP-T5 mutagenesis vector is provided, referenced herein as SEQ ID NO: 113. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and hsAID (K10E/E156G/T821).

FIG. 97. MP-T6 Vector Map. A schematic depiction of one embodiment of a MP-T6 mutagenesis vector is provided, referenced herein as SEQ ID NO: 114. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and hsAID (K10E/E156G/T821).

FIG. 98. MP-U Vector Map. A schematic depiction of one embodiment of a MP-U mutagenesis vector is provided, referenced herein as SEQ ID NO: 115. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and pmCDA1.

FIG. 99. MP-U2 Vector Map. A schematic depiction of one embodiment of a MP-U2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 116. This embodiment comprises araC, dnaQ926 RBS+, dam, seqA, emrR, ugi and pmCDA1.

FIG. 100. MP-U3 Vector Map. A schematic depiction of one embodiment of a MP-U3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 117. This embodiment comprises araC, dnaQ926, dam, seqA RBS+, emrR, ugi and pmCDA1.

FIG. 101. MP-U4 Vector Map. A schematic depiction of one embodiment of a MP-U4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 118. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi RBS+ and pmCDA1.

FIG. 102. MP-V Vector Map. A schematic depiction of one embodiment of a MP-V mutagenesis vector is provided, referenced herein as SEQ ID NO: 119. This embodiment comprises araC, dnaQ-BRM1, dam, seqA, emrR, ugi and pmCDA1.

FIG. 103. MP-V2 Vector Map. A schematic depiction of one embodiment of a MP-V2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 120. This embodiment comprises araC, dnaQ-BRM1, dam, seqA, emrR, ugi and pmCDA1.

FIG. 104. MP-V3 Vector Map. A schematic depiction of one embodiment of a MP-V3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 121. This embodiment comprises araC, dnaQ-BR11, dam, seqA, emrR, ugi and pmCDA1.

FIG. 105. MP-V4 Vector Map. A schematic depiction of one embodiment of a MP-V4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 122. This embodiment comprises araC, dnaQ-BR6, dam, seqA, emrR, ugi and pmCDA1.

FIG. 106. MP-V5 Vector Map. A schematic depiction of one embodiment of a MP-V5 mutagenesis vector is provided, referenced herein as SEQ ID NO: 123. This embodiment comprises araC, dnaQ-BR1, dam, seqA, emrR, ugi and pmCDA1.

 DEFINITIONS

The term “accessory plasmid,” as used herein, refers to a plasmid comprising a gene required for the generation of infectious viral particles under the control of a conditional promoter. In the context of continuous evolution of genes, transcription from the conditional promoter of the accessory plasmid is typically activated, directly or indirectly, by a function of the gene to be evolved. Accordingly, the accessory plasmid serves the function of conveying a competitive advantage to those viral vectors in a given population of viral vectors that carry a version of the gene to be evolved able to activate the conditional promoter or able to activate the conditional promoter more strongly than other versions of the gene to be evolved. In some embodiments, only viral vectors carrying an “activating” version of the gene to be evolved will be able to induce expression of the gene required to generate infectious viral particles in the host cell, and, thus, allow for packaging and propagation of the viral genome in the flow of host cells. Vectors carrying non-activating versions of the gene to be evolved, on the other hand, will not induce expression of the gene required to generate infectious viral vectors, and, thus, will not be packaged into viral particles that can infect fresh host cells.

The term “cellstat,” as used herein, refers to a culture vessel comprising host cells, in which the number of cells is substantially constant over time.

The term “continuous evolution,” as used herein, refers to an evolution process, in which a population of nucleic acids encoding a gene to be evolved is subjected to multiple rounds of (a) replication, (b) mutation, and (c) selection to produce a desired evolved version of the gene to be evolved that is different from the original version of the gene, for example, in that a gene product, such as, e.g., an RNA or protein encoded by the gene, exhibits a new activity not present in the original version of the gene product, or in that an activity of a gene product encoded by the original gene to be evolved is modulated (increased or decreased). The multiple rounds can be performed without investigator intervention, and the steps (a)-(c) can be carried out simultaneously. Typically, the evolution procedure is carried out in vitro, for example, using cells in culture as host cells. In general, a continuous evolution process provided herein relies on a system in which a gene encoding a gene product of interest is provided in a nucleic acid vector that undergoes a life-cycle including replication in a host cell and transfer to another host cell, wherein a critical component of the life-cycle is deactivated and reactivation of the component is dependent upon an activity of the gene to be evolved that is a result of a mutation in the nucleic acid vector.

The term “flow”, as used herein in the context of host cells, refers to a stream of host cells, wherein fresh host cells not harboring the transfer vector (e.g., the viral vector encoding the gene to be evolved) are being introduced into a host cell population, for example, a host cell population in a lagoon, remain within the population for a limited time, and are then removed from the host cell population. In a simple form, a host cell flow may be a flow through a tube, or a channel, for example, at a controlled rate. In other embodiments, a flow of host cells is directed through a lagoon that holds a volume of cell culture media and comprises an inflow and an outflow. The introduction of fresh host cells may be continuous or intermittent and removal may be passive, e.g., by overflow, or active, e.g., by active siphoning or pumping. Removal further may be random, for example, if a stirred suspension culture of host cells is provided, removed liquid culture media will contain freshly introduced host cells as well as cells that have been a member of the host cell population within the lagoon for some time. Even though, in theory, a cell could escape removal from the lagoon indefinitely, the average host cell will remain only for a limited period of time within the lagoon, which is determined mainly by the flow rate of the culture media (and suspended cells) through the lagoon. Since the viral vectors replicate in a flow of host cells, in which fresh, uninfected host cells are provided while infected cells are removed, multiple consecutive viral life cycles can occur without investigator interaction, which allows for the accumulation of multiple advantageous mutations in a single evolution experiment.

The term “fresh,” as used herein in the context of host cells, and used interchangeably with the terms “non-infected” or “uninfected” in the context of host cells of viral vectors, refers to a host cell that does not harbor the vector or, in the context of viral vectors, has not been infected by the viral vector comprising a gene to be evolved as used in a continuous evolution process provided herein. A fresh host cell can, however, have been infected by a viral vector unrelated to the vector to be evolved or by a vector of the same or a similar type but not carrying the gene of interest.

The term “gene of interest” or “gene to be evolved,” as used herein, refers to a nucleic acid construct comprising a nucleotide sequence encoding a gene product, e.g., an RNA or a protein, to be evolved in a continuous evolution process as provided herein. The term includes any variations of a gene of interest that are the result of a continuous evolution process according to methods provided herein. For example, in some embodiments, a gene of interest is a nucleic acid construct comprising a nucleotide sequence encoding an RNA or protein to be evolved, cloned into a viral vector, for example, a phage genome, so that the expression of the encoding sequence is under the control of one or more promoters in the viral genome. In other embodiments, a gene of interest is a nucleic acid construct comprising a nucleotide sequence encoding an RNA or protein to be evolved and a promoter operably linked to the encoding sequence. When cloned into a viral vector, for example, a phage genome, the expression of the encoding sequence of such genes of interest is under the control of the heterologous promoter and, in some embodiments, may also be influenced by one or more promoters comprised in the viral genome. In some embodiments, the term “gene of interest” or “gene to be evolved refers to a nucleic acid sequence encoding a gene product to be evolved, without any additional sequences. In some embodiments, the term also embraces additional sequences associated with the encoding sequence, such as, for example, intron, promoter, enhancer, or polyadenylation signal sequences.

The term “helper phage,” as used herein interchangeable with the terms “helper phagemid” and “helper plasmid,” refers to a nucleic acid construct comprising a phage gene required for the phage life cycle, or a plurality of such genes, but lacking a structural element required for genome packaging into a phage particle. For example, a helper phage may provide a wild-type phage genome lacking a phage origin of replication. In some embodiments, a helper phage is provided that comprises a gene required for the generation of phage particles, but lacks a gene required for the generation of infectious particles, for example, a full-length pIII gene. In some embodiments, the helper phage provides only some, but not all, genes for the generation of infectious phage particles. Helper phages are useful to allow modified phages that lack a gene for the generation of infectious phage particles to complete the phage life cycle in a host cell. Typically, a helper phage will comprise the genes for the generation of infectious phage particles that are lacking in the phage genome, thus complementing the phage genome. In the continuous evolution context, the helper phage typically complements the selection phage, but both lack a phage gene required for the production of infectious phage particles.

The terms “high copy number plasmid” and “low copy number plasmid” are art-recognized, and those of skill in the art will be able to ascertain whether a given plasmid is a high or low copy number plasmid. In some embodiments, a low copy number accessory plasmid is a plasmid exhibiting an average copy number of plasmid per host cell in a host cell population of about 5 to about 100. In some embodiments, a very low copy number accessory plasmid is a plasmid exhibiting an average copy number of plasmid per host cell in a host cell population of about 1 to about 10. In some embodiments, a very low copy number accessory plasmid is a single-copy per cell plasmid. In some embodiments, a high copy number accessory plasmid is a plasmid exhibiting an average copy number of plasmid per host cell in a host cell population of about 100 to about 5000.

The term “host cell,” as used herein, refers to a cell that can host, replicate, and transfer a phage vector useful for a continuous evolution process as provided herein. In embodiments where the vector is a viral vector, a suitable host cell is a cell that can be infected by the viral vector, can replicate it, and can package it into viral particles that can infect fresh host cells. A cell can host a viral vector if it supports expression of genes of viral vector, replication of the viral genome, and/or the generation of viral particles. One criterion to determine whether a cell is a suitable host cell for a given viral vector is to determine whether the cell can support the viral life cycle of a wild-type viral genome that the viral vector is derived from. For example, if the viral vector is a modified M13 phage genome, as provided in some embodiments described herein, then a suitable host cell would be any cell that can support the wild-type M13 phage life cycle. Suitable host cells for viral vectors useful in continuous evolution processes are well known to those of skill in the art, and the disclosure is not limited in this respect.

The term “infectious viral particle,” as used herein, refers to a viral particle able to transport the viral genome it comprises into a suitable host cell. Not all viral particles are able to transfer the viral genome to a suitable host cell. Particles unable to accomplish this are referred to as non-infectious viral particles. In some embodiments, a viral particle comprises a plurality of different coat proteins, wherein one or some of the coat proteins can be omitted without compromising the structure of the viral particle. In some embodiments, a viral particle is provided in which at least one coat protein cannot be omitted without the loss of infectivity. If a viral particle lacks a protein that confers infectivity, the viral particle is not infectious. For example, an M13 phage particle that comprises a phage genome packaged in a coat of phage proteins (e.g., pVIII) but lacks pIII (protein III) is a non-infectious M13 phage particle because pIII is essential for the infectious properties of M13 phage particles.

The term “lagoon.” as used herein, refers to a culture vessel or bioreactor through which a flow of host cells is directed. When used for a continuous evolution process as provided herein, a lagoon typically holds a population of host cells and a population of viral vectors replicating within the host cell population, wherein the lagoon comprises an outflow through which host cells are removed from the lagoon and an inflow through which fresh host cells are introduced into the lagoon, thus replenishing the host cell population.

The term “mutagen,” as used herein, refers to an agent that induces mutations or increases the rate of mutation in a given biological system, for example, a host cell, to a level above the naturally occurring level of mutation in that system. Some exemplary mutagens useful for continuous evolution procedures are provided elsewhere herein, and other useful mutagens will be evident to those of skill in the art. Useful mutagens include, but are not limited to, ionizing radiation, ultraviolet radiation, base analogs, deaminating agents (e.g., nitrous acid), intercalating agents (e.g., ethidium bromide), alkylating agents (e.g., ethylnitrosourea), transposons, bromine, azide salts, psoralen, benzene,3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) (CAS no. 77439-76-0), O,O-dimethyl-S-(phthalimidomethyl)phosphorodithioate (phos-met) (CAS no. 732-11-6), formaldehyde (CAS no. 50-00-0), 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) (CAS no. 3688-53-7), glyoxal (CAS no. 107-22-2), 6-mercaptopurine (CAS no. 50-44-2), N-(trichloromethylthio)-4-cyclohexane-1,2-dicarboximide (captan) (CAS no. 133-06-2), 2-aminopurine (CAS no. 452-06-2), methyl methane sulfonate (MMS) (CAS No. 66-27-3), 4-nitroquinoline 1-oxide (4-NQO) (CAS No. 56-57-5), N4-Aminocytidine (CAS no. 57294-74-3), sodium azide (CAS no. 26628-22-8), N-ethyl-N-nitrosourea (ENU) (CAS no. 759-73-9), N-methyl-N-nitrosourea (MNU) (CAS no. 820-60-0), 5-azacytidine (CAS no. 320-67-2), cumene hydroperoxide (CHP) (CAS no. 80-15-9), ethyl methanesulfonate (EMS) (CAS no. 62-50-0), N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) (CAS no. 4245-77-6), N-methyl-N-nitro-N-nitrosoguanidine (MNNG) (CAS no. 70-25-7), 5-diazouracil (CAS no. 2435-76-9), and t-butyl hydroperoxide (BHP) (CAS no. 75-91-2). Additional mutagens can be used in continuous evolution procedures as provided herein, and the invention is not limited in this respect.

The term “mutagenesis plasmid,” as used herein, refers to a plasmid comprising a nucleic acid sequence encoding a gene product or a combination of gene products that act(s) as a mutagen.

The term “nucleic acid,” as used herein, refers to a polymer of nucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1-methyl adenosine, 1-methyl guanosine, N6-methyl adenosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, 2′-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

The term “phage,” as used herein interchangeably with the term “bacteriophage,” refers to a virus that infects bacterial cells. Typically, phages consist of an outer protein capsid enclosing genetic material. The genetic material can be ssRNA, dsRNA, ssDNA, or dsDNA, in either linear or circular form. Phages and phage vectors are well known to those of skill in the art and non-limiting examples of phages that are useful for carrying out the methods provided herein are λ (Lysogen), T2, T4, T7, T12, R17, M13, MS2, G4, P1, P2, P4, Phi X174, N4, Φ6, and Φ29. In certain embodiments, the phage utilized in the present invention is M13. Additional suitable phages and host cells will be apparent to those of skill in the art and the invention is not limited in this aspect. For an exemplary description of additional suitable phages and host cells, see Elizabeth Kutter and Alexander Sulakvelidze: Bacteriophages: Biology and Applications. CRC Press; 1st edition (December 2004), ISBN: 0849313368; Martha R. J. Clokie and Andrew M. Kropinski: Bacteriophages; Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions (Methods in Molecular Biology) Humana Press; 1st edition (December, 2008), ISBN: 1588296822; Martha R. J. Clokie and Andrew M. Kropinski: Bacteriophages: Methods and Protocols, Volume 2: Molecular and Applied Aspects (Methods in Molecular Biology) Humana Press; 1st edition (December 2008), ISBN: 1603275649; all of which are incorporated herein in their entirety by reference for disclosure of suitable phages and host cells as well as methods and protocols for isolation, culture, and manipulation of such phages).

The term “phage-assisted continuous evolution (PACE),” as used herein, refers to continuous evolution that employs phage as viral vectors. PACE technology has been described previously, for example, in International PCT Application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. application, U.S. Ser. No. 13/922,812, filed Jun. 20, 2013; and U.S. application, U.S. Ser. No. 62/067,194, filed Oct. 22, 2014, each of which is incorporated herein by reference.

The term “promoter” is art-recognized and refers to a nucleic acid molecule with a sequence recognized by the cellular transcription machinery and able to initiate transcription of a downstream gene. A promoter can be constitutively active, meaning that the promoter is always active in a given cellular context, or conditionally active, meaning that the promoter is only active in the presence of a specific condition. For example, a conditional promoter may only be active in the presence of a specific protein that connects a protein associated with a regulatory element in the promoter to the basic transcriptional machinery, or only in the absence of an inhibitory molecule. A subclass of conditionally active promoters are inducible promoters that require the presence of a small molecule “inducer” for activity. Examples of inducible promoters include, but are not limited to, arabinose-inducible promoters, Tet-on promoters, and tamoxifen-inducible promoters. A variety of constitutive, conditional, and inducible promoters are well known to the skilled artisan, and the skilled artisan will be able to ascertain a variety of such promoters useful in carrying out the instant invention, which is not limited in this respect.

The term “protein,” as used herein refers to a polymer of amino acid residues linked together by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptide of any size, structure, or function. Typically, a protein will be at least three amino acids long. A protein may refer to an individual protein or a collection of proteins. Inventive proteins preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain; see, for example, cco.caltech.edu/˜dadgrp/Unnatstruct.gif, which displays structures of non-natural amino acids that have been successfully incorporated into functional ion channels) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in an inventive protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein may also be a single molecule or may be a multi-molecular complex. A protein may be just a fragment of a naturally occurring protein or peptide. A protein may be naturally occurring, recombinant, or synthetic, or any combination of these.

The term “replication product,” as used herein, refers to a nucleic acid that is the result of viral genome replication by a host cell. This includes any viral genomes synthesized by the host cell from a viral genome inserted into the host cell. The term includes non-mutated as well as mutated replication products.

The term “selection phage,” as used herein interchangeably with the term “selection plasmid,” refers to a modified phage that comprises a gene of interest to be evolved and lacks a full-length gene encoding a protein required for the generation of infectious phage particles. For example, some M13 selection phages provided herein comprise a nucleic acid sequence encoding a gene to be evolved, e.g., under the control of an M13 promoter, and lack all or part of a phage gene encoding a protein required for the generation of infectious phage particles, e.g., gI, gII, gIII, gIV, gV, gVI, gVII, gVIII, gIX, or gX, or any combination thereof. For example, some M13 selection phages provided herein comprise a nucleic acid sequence encoding a gene to be evolved, e.g., under the control of an M13 promoter, and lack all or part of a gene encoding a protein required for the generation of infective phage particles, e.g., the gIII gene encoding the pIII protein.

The terms “small molecule” and “organic compound” are used interchangeably herein and refer to molecules, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight. Typically, an organic compound contains carbon. An organic compound may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyl, carbonyls, or heterocyclic rings). In some embodiments, organic compounds are monomeric and have a molecular weight of less than about 1500 g/mol. In certain embodiments, the molecular weight of the small molecule is less than about 1000 g/mol or less than about 500 g/mol. In certain embodiments, the small molecule is a therapeutic drug or drug candidate, for example, a drug or drug candidate that is in clinical or pre-clinical trials or that has already been deemed safe and effective for use in humans or animals by the appropriate governmental agency or regulatory body.

The term “turbidostat,” as used herein, refers to a culture vessel comprising host cells in suspension culture, in which the turbidity of the culture medium is substantially essentially constant over time. In some embodiments, the turbidity of a suspension culture, for example, of bacterial cells, is a measure for the cell density in the culture medium. In some embodiments, a turbidostat comprises an inflow of fresh media and an outflow, and a controller that regulates the flow into and/or out of the turbidostat based on the turbidity of the suspension culture in the turbidostat.

The term “vector,” as used herein, refers to a nucleic acid that can be modified to encode a gene of interest and that is able to enter into a host cell, mutate and replicate within the host cell, and then transfer a replicated form of the vector into another host cell. Exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages, and conjugative plasmids. Additional suitable vectors will be apparent to those of skill in the art based on the instant disclosure.

The term “viral life cycle,” as used herein, refers to the viral reproduction cycle comprising insertion of the viral genome into a host cell, replication of the viral genome in the host cell, and packaging of a replication product of the viral genome into a viral particle by the host cell.

The term “viral particle,” as used herein, refers to a viral genome, for example, a DNA or RNA genome, that is associated with a coat of a viral protein or proteins, and, in some cases, with an envelope of lipids. For example, a phage particle comprises a phage genome packaged into a protein encoded by the wild type phage genome.

The term “viral vector.” as used herein, refers to a nucleic acid comprising a viral genome that, when introduced into a suitable host cell, can be replicated and packaged into viral particles able to transfer the viral genome into another host cell. The term viral vector extends to vectors comprising truncated or partial viral genomes. For example, in some embodiments, a viral vector is provided that lacks a gene encoding a protein essential for the generation of infectious viral particles. In suitable host cells, for example, host cells comprising the lacking gene under the control of a conditional promoter, however, such truncated viral vectors can replicate and generate viral particles able to transfer the truncated viral genome into another host cell. In some embodiments, the viral vector is a phage, for example, a filamentous phage (e.g., an M13 phage). In some embodiments, a viral vector, for example, a phage vector, is provided that comprises a gene of interest to be evolved.

DETAILED DESCRIPTION Introduction

Some aspects of this disclosure provide systems, vectors, and methods for modulating the mutation rates in cells, for example, in bacterial host cells. In some embodiments, the present disclosure provides mutagenesis vectors, sometimes referred to herein as mutagenesis expression constructs or, if in the form of a plasmid, as mutagenesis plasmids, that mediate highly potent, broad-spectrum, and controllable mutagenesis in bacterial cells. These vectors can be used in many bacterial strains, including, but not limited to, most E. coli strains, to mutagenize chromosomal, episomal, or viral DNA, enabling high-efficiency mutation, for example, during directed evolution, obviating the need to create random DNA libraries in vitro, and bypassing transformation efficiency bottlenecks. Some advantages of the systems, vectors, and methods for modulating the mutation rates in bacteria as provided herein include, for example, the ability to evolve a gene of interest in a significantly reduced time frame as compared to other mutagenic technologies, and the fact that the mutagenesis-inducing vectors and constructs are not harmful to humans, in contrast to many other mutagens (e.g., chemical mutagens, UV, or ionizing radiation) commonly used. The utility and the improved mutation rate of this system is demonstrated herein by evolving resistance to eight antibiotics in bacterial cells significantly more effectively than when using several other widely used mutagenesis methods. The mutagenesis efficiency conferred by the systems, vectors, and methods provided herein is exemplified herein by an exemplary use of some embodiments of these systems, vectors, and methods to evolve RNA polymerase specificity in bacteriophage under conditions that previously necessitated the use of mutational drift or evolutionary stepping-stones.

Access to new mutations drives both natural and laboratory evolution. Native biological mutation rates, sometimes referred to herein as “basal mutation rates,” are modest, occurring at frequencies of approximately one mutation per billion replicated DNA bases in most eukaryotes and prokaryotes [1]. Under such native mutation conditions, the time to evolve gene variants that improve organismal fitness is strongly determined by the time required for the mutation to appear [2]. Laboratory evolution methods typically increase basal mutation rates to accelerate the discovery of biomolecules with desired properties on a practical time scale. In addition to mutation rate, the mutational spectrum is also a crucial component of the genetic diversity that fuels evolution. Access to diverse amino acid substitutions during evolution is enhanced by more complete coverage of the 12 possible mutation types [3].

Several systems to enhance mutational efficiency and broaden mutational spectra have been developed for use in laboratory evolution efforts [4]. In vitro mutagenesis through error-prone PCR, site-saturation mutagenesis, or DNA shuffling has become the standard approach to introduce diversity into genes of interest. Whereas in vitro mutagenesis methods allow for control of mutation rate and mutational spectrum, in vivo mutagenesis methods allow for mutation and selection cycles to be coupled, bypass transformation efficiency bottlenecks that frequently limit the size of gene populations that can be accessed following in vitro mutagenesis, and avoid labor-intensive library creation, cloning, and manipulation steps [5]. The difficulty of tuning mutagenic load and spectrum in live cells, however, has prevented the development of safe, effective in vivo mutagenesis methods that can rival or exceed the efficiency and mutational spectra of state-of-the-art in vitro methods.

The most commonly used in vivo mutagenesis methods include chemical mutagens, nucleobase analogs, UV light, and hypermutator strains [3]. Chemical mutagens yield narrow mutagenic spectra and are potent human carcinogens. Base analogs offer a safer alternative to chemical mutagenesis, but also exhibit narrow mutational spectra. UV radiation is known to generate a wide mutation spectrum with little sequence preference, but with low potency that is limited by the toxicity of high doses of UV radiation.

Perhaps the most widespread method for in vivo mutagenesis has been the use of hypermutator strains such as XL-1 Red [6] that have been engineered to have higher mutation rates through the deletion or modification of genes involved in DNA replication and repair. These strains suffer from numerous drawbacks, however, including modest mutational potency (vide infra), moderately biased mutational preference [7], poor transformation efficiency, slow growth rate, and difficulty of modification when additional genetic manipulations are required. In addition, the rate of mutagenesis is not controllable using common hypermutator strains, and mutagenesis generally must be separated from other steps such as selection or screening due to the instability or poor growth of the strain. Loeb and coworkers previously described an elegant system that uses a low-fidelity E. coli DNA Pol I (LF-Pol I) to increase in vivo mutagenesis efficiency with a wide scope of mutational types [8]. Unfortunately, this method is restricted to Pol I temperature-sensitive strains and mutates only vectors carrying ColE1-related origins of replication, with mutation rate being highly dependent on distance from the ColE1 origin [8, 9].

An in vivo mutagenesis method should offer one or more of the following: (i) a broad mutagenesis spectrum, (ii) a mutation rate that can be very high but is easily modulated by the researcher, and (iii) entirely episomal encoding so that it can be applied to virtually any bacterial strain, including, but not limited to, virtually any E. coli strain. Some aspects of this disclosure describe the development of general in vivo mutagenesis systems, vectors, and method that meet these criteria.

Mutagenesis Constructs

Some aspects of this disclosure provide expression constructs encoding gene products that increase the mutation rate in a host cell, e.g., in a bacterial host cell. Expression constructs are sometimes also referred to as vectors. In some embodiments, the expression constructs are plasmids, also referred to herein as mutagenesis plasmids. The use of mutagenesis plasmids in the context of directed evolution has previously been described (e.g., in International PCT Application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. application, U.S. Ser. No. 13/922,812, filed Jun. 20, 2013; and U.S. application, U.S. Ser. No. 62/067,194, filed Oct. 22, 2014, the entire contents of each of which are incorporated herein by reference). Some aspects of this disclosure provide improved mutagenesis systems and expression constructs using various combinations of mutagenesis-inducing gene products. The expression constructs provided herein can be used to induce mutagenesis in a target cell, e.g., in a bacterial host cell, at increased rates as compared to conventional mutagenesis agents and methods.

Some aspects of this disclosure provide expression constructs for modulating the mutation rate of nucleic acids in a cell, e.g., in a bacterial, yeast, or eukaryotic cell. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a gene product that disrupts a proof-reading pathway, a translesion synthesis pathway, a methyl-directed mismatch repair pathway, a base excision repair pathway, or a base selection pathway of the bacterial cell, or any combination thereof, wherein the nucleic acid sequence encoding the gene product is under the control of a heterologous promoter.

Some aspects of this disclosure provide expression constructs encoding a combination of mutagenesis-inducing gene products that allow for robust DNA mutagenesis, e.g., robust chromosomal, episomal, and viral DNA mutagenesis in host cells, e.g., in bacterial host cells. Some exemplary, non-limiting, combinations of mutagenesis-inducing gene products are listed in Table 2 or FIG. 7. The nomenclature of the nucleotides (e.g., genes, transcripts) and proteins referred to herein adheres to the official nomenclature used by the National Center for Biotechnology Information (NCBI). Nucleotide and protein sequences related to the gene symbols listed herein, e.g., in Table 2 or FIG. 7, are known to those of skill in the art. For gene symbols for which publications are indicated (e.g., by superscript numbers or in square brackets), any sequences provided in the publication related to the gene symbol is incorporated herein by reference. In some embodiments, the gene symbols above relate to nucleotide and protein sequences accessible under that gene symbol in any of the National Center for Biotechnology Information (NCBI) databases, for example, in the Nucleotide Reference Sequence (RefSeq) database, release 69 (Jan. 2, 2015), or in the Unigene database available at the time of filing, and any NCBI database entries, e.g., RefSeq database entries for the listed genes in the ResSeq database, release 69, or in the Unigene database at the time of filing, are incorporated herein by reference.

In some embodiments, a plurality of nucleic acid sequences encoding a gene product that increases the mutation rate in a cell are employed, e.g., combinations of such gene products as shown in Table 2 or FIG. 7. For example, in some embodiments, an a mutagenesis construct provided herein comprises at least two, at least three, at least four, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different nucleic acid sequences encoding a different gene product, e.g., a gene product of Table 2 or FIG. 7. For example, in some embodiments, a mutagenesis expression construct as provided herein may include at least a combination of sequences encoding dnaQ92640, umuD′, umuC, and recA730, and at least one, at least two, at least three, at least four, or at least five additional nucleic acid sequence encoding a different gene product listed in Table 2 or FIG. 7. In some embodiments, a mutagenesis expression construct as provided herein may include at least a combination of sequences encoding dnaQ926, dam, and seqA, and at least one, at least two, at least three, at least four, or at least five additional nucleic acid sequence encoding a different gene product listed in Table 2 or FIG. 7.

It will be understood that, in some embodiments, the expression constructs provided herein include wild-type sequences of the genes or encoding sequences provided herein, e.g., in Table 2 or FIG. 7, while in other embodiments, one or more of the genes or encoding sequences used may be a functional variant, mutant, fragment, or recombinant form of a wild-type sequence. Such functional variants, mutants, fragments, or recombinant forms include sequences with silent mutations, e.g., codon-optimized sequences, in which nucleotides have been exchanged without altering the sequence of an encoded gene product (e.g., an encoded protein), sequences comprising naturally-occurring polymorphisms, truncations of wild-type sequences that retain the function of the wild-type sequence (e.g., 5′- or 3′ UTR truncations, intron deletions (e.g., cDNAs), and deletions of domains not required for gene product function, and fusions with other sequences, including, e.g., sequences encoding a tag or other gene product. Suitable variants, mutants, fragments, or recombinant forms of wild-type sequences provided herein will be apparent to the skilled artisan based on the instant disclosure.

In some embodiment, the present disclosure provides expression constructs for modulating the mutagenesis rate in bacterial cells harboring such constructs, also referred to herein as host cells. Such expression constructs can be configured in any suitable manner for introduction into and expression in a bacterial cell, e.g., in the form of a plasmid, cosmid, bacteriophage, or artificial bacterial chromosome. In embodiments, where the expression construct is configured as or comprised in a plasmid, such plasmids are also sometimes referred to herein as “mutagenesis plasmids.” In some embodiments, the bacterial expression constructs provided herein allow for modulation of mutagenesis in bacterial host cells in a strain-independent manner.

In some embodiments, an expression construct for modulating the mutagenesis rate in a bacterial host cell as provided herein may encode a DNA polymerase lacking a proofreading capability. In some embodiments, the expression construct may encode a gene product involved in the bacterial SOS stress response, for example, a component of a bacterial translesion synthesis polymerase V. In some embodiments, the expression construct may encode a deoxyadenosine methylase. In some embodiments, the expression construct may encode a hemimethylated-GATC binding domain. In some non-limiting embodiments, the expression construct encodes UmuC (a component of E. coli translesion synthesis polymerase V), dam (deoxyadenosine methylase), and/or seqA (hemimethylated-GATC binding domain), or any combination thereof.

In some embodiments, the expression construct for modulating the mutagenesis rate in a bacterial host cell comprises a nucleic acid sequence encoding a gene product that disrupts a proofreading pathway, a translesion synthesis pathway, a methyl-directed mismatch repair pathway, a base excision repair pathway, or a base selection pathway of the bacterial cell, or any combination thereof, wherein the nucleic acid sequence encoding the gene product is under the control of a heterologous promoter.

In some embodiments, the gene product that disrupts a proofreading pathway is a dnaQ926, BRM1, BR11, BR1, BR6, or BR13 gene product. In some embodiments, the gene product that disrupts a translesion synthesis pathway is an umuD′, umuC, recA, dinB, or polB gene product.

In some embodiments, the gene product that disrupts a translesion synthesis pathway is an umuD′, umuC, recA, dinB, or polB gene product. In some embodiments, the recA gene product is a recA730 gene product. In some embodiments, the polB gene product is a polB(D156A) gene product. In some embodiments, the gene product that disrupts a methyl-directed mismatch repair pathway is a mutS, mutL, mutH, dam, or seqA gene product. In some embodiments, the mutS gene product is a mutS538, mutS503, or mutSΔN gene product. In some embodiments, the mutL gene product is a mutL705, mutL713, mutL(R261H), or mutL(K307A) gene product. In some embodiments, the mutH gene product is a mutH(E56A), mutH(K79E), or mutH(K116E) gene product. In some embodiments, the gene product that disrupts a base excision repair pathway is a ugi, AID, APOBEC, CDA, MAG, or AAG gene product. In some embodiments, the AID gene product is an AID (7), AID (7.3), AID (7.3.5), AID (7.3.3), AID (7.3.1), or AID (7.3.2) gene product. In some embodiments, the APOBEC gene product is an APOBEC 1 gene product. In some embodiments, the CDA gene product is a CDA1 gene product. In some embodiments, the MAG gene product is a MAG1 gene product. In some embodiments, the AAG gene product is an AAG(Y127I-H136L) or Δ80-AAG(Y127I-H136L) gene product.

In some embodiments, the gene product that disrupts a base selection pathway is a dnaE74, dnaE486, dnaE1026, dnaX36, dnaX2016, emrR, nrdAB, nrdA(H59A)B, nrdA(A65V)B, nrdA(A301V)B, nrdAB(P334L), or nrdEF gene product.

In some embodiments, the expression construct comprises a nucleic acid sequence encoding a rsmE, cchA, yffI, or yfjY gene product.

In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaQ926 gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a umuD′, umuC, or recA730 gene product, or any combination thereof. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a dnaE486, dnaE1026, dnaX36, or dnaX2016 gene product, or any combination thereof. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a mutS538, mutS503, mutL705, mutL713, mutL(R261H), mutL4K307A), mutH(E56A), mutH(K79E), or mutH(KI 16E) gene product, or any combination thereof. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a Dam gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a seqA gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a emrR, mutH(E56A), mutL713, mutS503, mutSΔN, dinB, or polB gene product, or any combination thereof.

In some embodiments, the expression construct comprises a nucleic acid sequence encoding a Dam gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a seqA gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a emrR gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a ugi gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a AID, AID (7), AID (7.3), AID (7.3.5), AID (7.3.3), AID (7.3.1), AID (7.3.2), APOBEC1, or CDA1 gene product, or any combination thereof. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a mutSΔN gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a rsmE, cchA, yffI, yfjY, nrdAB, nrdA(H59A)B, nrdA(A65V)B, nrdA(A301V)B, nrdEF, or nrdAB(P334L)gene product, or any combination thereof.

In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaE74 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaE486 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaE1026 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaX36 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaX2016 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a rpsD12 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a rpsD14 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a rpsD16 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a polB gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a polB(D156A) gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a MAG1 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a AAG(Y127I-H136L) gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a Δ80-AAG(Y127I-H136L) gene product.

In some embodiments, the expression construct is comprised in a plasmid. In some embodiments, the plasmid comprises a bacterial origin of replication. In some embodiments, the origin of replication is a cloDF13 origin of replication. In some embodiments, the plasmid comprises a nucleic acid sequence encoding a gene product conferring resistance to an antibiotic to a bacterial host cell. In some embodiments, the antibiotic is chloramphenicol, kanamycin, tetracycline, or ampicillin.

In some embodiments, the expression construct comprises at least one inducible promoter controlling the expression of at least one nucleic acid sequence encoding the gene product that disrupts a proofreading pathway, a translesion synthesis pathway, a methyl-directed mismatch repair pathway, a base excision repair pathway, or a base selection pathway, or any combination thereof. In some embodiments, the inducible promoter controls the expression of a nucleic acid encoding a combination of two or more mutagenic gene products, e.g., of any combination listed in Table 2 or FIG. 7. In some embodiments, the expression construct comprises an inducible promoter controlling expression of a nucleic acid sequence encoding a component of E. coli translesion synthesis polymerase V, a deoxyadenosine methylase, and/or a hemimethylated-GATC binding domain, or any combination thereof. In some embodiments, the component of E. coli translesion synthesis polymerase V is umuC. In some embodiments, the deoxyadenosine methylase is dam. In some embodiments, the hemimethylated-GATC binding domain is seqA. In some embodiments, the nucleic acid sequence controlled by the inducible promoter encodes at least one, at least two, at least three, or at least four additional mutagenic gene products, e.g., as listed in Table 2 or FIG. 7. In some embodiments, the inducible promoter is an arabinose responsive promoter. In some embodiments, the arabinose responsive promoter is a PBAD promoter. In some embodiments, the expression construct comprises a nucleic acid sequence encoding an arabinose operon regulatory protein. In some embodiments, the arabinose operon regulatory protein is araC. In some embodiments, the nucleic acid sequence encoding the arabinose operon regulatory protein is under the control of a weak promoter. In some embodiments, the weak promoter is a PC promoter.

In some embodiments, the expression construct comprises at least one codon-optimized nucleic acid sequence encoding a gene product. Methods for codon-optimization and codons preferred by various types of bacterial host cells are well known to those of skill in the art. Some exemplary suitable methods and codons are provided herein, and additional methods and codons will be apparent to the skilled artisan based on the present disclosure. In some embodiments, the codon-optimized nucleic acid sequence comprises at least one codon of a naturally-occurring sequence encoding the gene product that has been replaced with a different codon encoding the same amino acid. In some embodiments, the at least one codon replacing the codon of the naturally occurring nucleic acid sequence corresponds to a tRNA expressed in a bacterial host cell, e.g., in an E. coli host cell. In some embodiments, the at least one codon replacing the codon of the naturally occurring nucleic acid sequence corresponds to a tRNA that is expressed at a higher abundance in the bacterial host cell than the tRNA relating to the naturally occurring tRNA. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a ribosome-binding site, wherein at least one ribosome-binding site encoded by the expression construct is modified as compared to a naturally occurring ribosome binding site. In some embodiments, the modified ribosome binding site exhibits increased ribosome binding as compared to the naturally occurring ribosome binding site. Ribosome-binding sites preferred by ribosomes in various host cells are well known to those of skill in the art.

In some embodiments, the expression construct comprises a nucleic acid sequence encoding a gene product or a combination of such sequences listed in Table 2 or FIG. 7.

Some aspects of this disclosure provide plasmids comprising an expression construct as disclosed herein.

Some aspects of this disclosure provide a cell comprising an expression construct or a plasmid as provided herein. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell further comprises a selection plasmid or an accessory plasmid. In some embodiments, the cell is a host cell for a bacteriophage. In some embodiments, the cell is an E. coli cell. In some embodiments, the cell is comprised in a lagoon.

Some aspects of this disclosure provide vectors and reagents for carrying out continuous evolution processes using the inventive mutagenesis constructs. Such vectors and reagents comprise, for example, selection phage, accessory plasmid, and helper plasmid vectors.

In some embodiments, a selection phage is provided that comprises a phage genome deficient in at least one gene required for the generation of infectious phage particles and a gene encoding a gene of interest to be evolved.

For example, in some embodiments, a selection phage as described in PCT Application PCT/US2009/056194, published as WO2010/028347 on Mar. 11, 2010; PCT Application PCT/US2011/066747, published as WO2012/088381 on Jun. 28, 2012; and U.S. application, U.S. Ser. No. 13/922,812, filed Jun. 20, 2013, the entire contents of each of which are incorporated herein by reference, is provided, that comprises a multiple cloning site for insertion of a nucleic acid sequence encoding a gene to be evolved of interest.

Such selection phage vectors typically comprise an M13 phage genome deficient in a gene required for the generation of infectious M13 phage particles, for example, a full-length gIII. In some embodiments, the selection phage comprises a phage genome providing all other phage functions required for the phage life cycle except the gene required for generation of infectious phage particles. In some such embodiments, an M13 selection phage is provided that comprises a gI, gII, gIV, gV, gVI, gVII, gVIII, gIX, and a gX gene, but not a full-length gIII. In some embodiments, the selection phage comprises a 3′-fragment of gIII, but no full-length gIII. The 3′-end of gIII comprises a promoter and retaining this promoter activity is beneficial, in some embodiments, for an increased expression of gVI, which is immediately downstream of the gIII 3′-promoter, or a more balanced (wild-type phage-like) ratio of expression levels of the phage genes in the host cell, which, in turn, can lead to more efficient phage production. In some embodiments, the 3′-fragment of gIII gene comprises the 3′-gIII promoter sequence. In some embodiments, the 3′-fragment of gIII comprises the last 180 bp, the last 150 bp, the last 125 bp, the last 100 bp, the last 50 bp, or the last 25 bp of gIII. In some embodiments, the 3′-fragment of gIII comprises the last 180 bp of gIII. In some embodiments, the multiple cloning site for insertion of the gene of interest is located downstream of the gVIII 3′-terminator and upstream of the gIII-3′-promoter.

Some aspects of this invention provide a vector system for continuous evolution procedures, comprising of a viral vector, for example, a selection phage, comprising a multiple cloning site for insertion of a gene to be evolved, a matching accessory plasmid, and a mutagenesis expression construct as described herein. In some embodiments, a vector system for phage-based continuous directed evolution is provided that comprises (a) a selection phage comprising a multiple cloning site for insertion of a gene of interest to be evolved, wherein the phage genome is deficient in at least one gene required to generate infectious phage; (b) an accessory plasmid comprising the at least one gene required to generate infectious phage particle under the control of a conditional promoter that is activated in response to a desired activity of the gene to be evolved; and (c) a mutagenesis expression construct as provided herein. In some embodiments, the mutagenesis expression construct comprises a nucleic acid sequence encoding a gene or combination of genes as listed in Table 2 or FIG. 7. In some embodiments, the mutagenesis expression construct is a mutagenesis plasmid.

In some embodiments, the selection phage is an M13 phage as described herein. For example, in some embodiments, the selection phage comprises an M13 genome including all genes required for the generation of phage particles, for example, gI, gII, gIV, gV, gVI, gVII, gVIII, gIX, and gX gene, but not a full-length gIII gene. In some embodiments, the selection phage genome comprises an F1 or an M13 origin of replication. In some embodiments, the selection phage genome comprises a 3′-fragment of gIII gene. In some embodiments, the selection phage comprises a multiple cloning site upstream of the gIII 3′-promoter and downstream of the gVIII 3′-terminator for insertion of a gene of interest.

The vector system may further comprise a helper phage, wherein the selection phage does not comprise all genes for the generation of infectious phage particles, and wherein the helper phage complements the genome of the selection phage, so that the helper phage genome and the selection phage genome together comprise at least one functional copy of all genes for the generation of phage particles, but are deficient in at least one gene required for the generation of infectious phage particles, which is provided by an accessory plasmid.

Methods

Some aspects of this disclosure provide methods for modulating the mutation rate in a cell, e.g., in a host cell for bacteriophage. In some embodiments, the method comprises contacting the cell with an expression construct or a plasmid as disclosed herein. In some embodiments, the expression construct comprises an inducible promoter, and the method further comprises culturing the host cell under conditions suitable to induce expression from the inducible promoter. In some embodiments, the inducible promoter is an arabinose-inducible promoter, and culturing the host cell under conditions suitable to induce expression from the inducible promoter comprises contacting the host cell with an amount of arabinose sufficient to increase expression of the arabinose-inducible promoter by at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, at least 10000-fold, at least 50000-fold, at least 100000-fold, at least 500000-fold, or at least 1000000-fold as compared to basal expression in the absence of arabinose. In some embodiments, the method results in an at least 10-fold, at least 100-fold, at least 1000-fold, at least 10000-fold, at least 15000-fold, at least 200000-fold, at least 250000-fold, or at least 300000-fold increased mutation rate as compared to the mutation rate in the host cell in the absence of the expression construct.

Some aspects of this disclosure provide methods for directed evolution using a mutagenesis expression constructs provided herein. In some embodiments, the method comprises (a) contacting a population of host cells comprising a mutagenesis expression construct or plasmid as provided herein with a population of phage vectors comprising a gene to be evolved and deficient in at least one gene for the generation of infectious phage particles, wherein (1) the host cells are amenable to transfer of the vector, (2) the vector allows for expression of the gene to be evolved in the host cell, can be replicated by the host cell, and the replicated vector can transfer into a second host cell; (3) the host cell expresses a gene product encoded by the at least one gene for the generation of infectious phage particles of (a) in response to the activity of the gene to be evolved, and the level of gene product expression depends on the activity of the gene to be evolved; (b) incubating the population of host cells under conditions allowing for mutation of the gene to be evolved and the transfer of the vector comprising the gene to be evolved from host cell to host cell, wherein host cells are removed from the host cell population, and the population of host cells is replenished with fresh host cells that comprise the expression construct but do not harbor the vector, and (c) isolating a replicated vector from the host cell population in (b), wherein the replicated vector comprises a mutated version of the gene to be evolved.

In some embodiments, the expression construct comprises an inducible promoter, wherein the incubating of (b) comprises culturing the population of host cells under conditions suitable to induce expression from the inducible promoter. In some embodiments, the inducible promoter is an arabinose-inducible promoter, wherein the incubating of (b) comprises contacting the host cell with an amount of arabinose sufficient to increase expression of the arabinose-inducible promoter by at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, at least 10000-fold, at least 50000-fold, at least 100000-fold, at least 500000-fold, or at least 1000000-fold as compared to basal expression in the absence of arabinose.

In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a phage. In some embodiments, the phage is a filamentous phage. In some embodiments, the phage is an M13 phage.

In some embodiments, the at least one gene for the generation of infectious phage particles comprises a sequence encoding a pIII protein. In some embodiments, the at least one gene for the generation of infectious phage particles comprises a full-length gIII gene. In some embodiments, the host cells comprise all phage genes except for the at least one gene for the generation of infectious phage particles in the form of a helper phage. In some embodiments, the phage genes comprised on the helper phage comprise pI, pII, pIV, pV, pVI, pVII, pVIII, pIX, and/or pX.

In some embodiments, the host cells comprise an accessory plasmid. In some embodiments, the accessory plasmid comprises an expression construct encoding the pIII protein under the control of a promoter that is activated by a gene product encoded by the gene to be evolved. In some embodiments, the host cells comprise the accessory plasmid and together, the helper phage and the accessory plasmid comprise all genes required for the generation of an infectious phage. In some embodiments, the method further comprises a negative selection for undesired activity of the gene to be evolved. In some embodiments, the host cells comprise an expression construct encoding a dominant-negative pIII protein (pIII-neg). In some embodiments, expression of the pIII-neg protein is driven by a promoter the activity of which depends on an undesired function of the gene to be evolved.

In some embodiments, step (b) comprises incubating the population of host cells for a time sufficient for at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least, 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 7500, at least 10000, or more consecutive life cycles of the viral vector or phage. In some embodiments, the host cells are E. coli cells.

In some embodiments, the host cells are incubated in suspension culture. In some embodiments, the population of host cells is continuously replenished with fresh host cells that do not comprise the vector. In some embodiments, fresh cells are being replenished and cells are being removed from the cell population at a rate resulting in a substantially constant number of cells in the cell population. In some embodiments, fresh cells are being replenished and cells are being removed from the cell population at a rate resulting in a substantially constant vector population. In some embodiments, fresh cells are being replenished and cells are being removed from the cell population at a rate resulting in a substantially constant vector, viral, or phage load. In some embodiments, the rate of fresh cell replenishment and/or the rate of cell removal is adjusted based on quantifying the cells in the cell population. In some embodiments, the rate of fresh cell replenishment and/or the rate of cell removal is adjusted based on quantifying the frequency of host cells harboring the vector and/or of host cells not harboring the vector in the cell population. In some embodiments, the quantifying is by measuring the turbidity of the host cell culture, measuring the host cell density, measuring the wet weight of host cells per culture volume, or by measuring light extinction of the host cell culture.

In some embodiments, the host cells are exposed to a mutagen. In some embodiments, the mutagen is ionizing radiation, ultraviolet radiation, base analogs, deaminating agents (e.g., nitrous acid), intercalating agents (e.g., ethidium bromide), alkylating agents (e.g., ethylnitrosourea), transposons, bromine, azide salts, psoralen, benzene,3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) (CAS no. 77439-76-0), 0,0-dimethyl-S-(phthalimidomethyl)phosphorodithioate (phos-met) (CAS no. 732-11-6), formaldehyde (CAS no. 50-00-0), 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) (CAS no. 3688-53-7), glyoxal (CAS no. 107-22-2), 6-mercaptopurine (CAS no. 50-44-2), N-(trichloromethylthio)-4-cyclohexane-1,2-dicarboximide (captan) (CAS no. 133-06-2), 2-aminopurine (CAS no. 452-06-2), methyl methane sulfonate (MMS) (CAS No. 66-27-3), 4-nitroquinoline 1-oxide (4-NQO) (CAS No. 56-57-5), N4-Aminocytidine (CAS no. 57294-74-3), sodium azide (CAS no. 26628-22-8), N-ethyl-N-nitrosourea (ENU) (CAS no. 759-73-9), N-methyl-N-nitrosourea (MNU) (CAS no. 820-60-0), 5-azacytidine (CAS no. 320-67-2), cumene hydroperoxide (CHP) (CAS no. 80-15-9), ethyl methanesulfonate (EMS) (CAS no. 62-50-0), N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) (CAS no. 4245-77-6), N-methyl-N-nitro-N-nitrosoguanidine (MNNG) (CAS no. 70-25-7), 5-diazouracil (CAS no. 2435-76-9), or t-butyl hydroperoxide (BHP) (CAS no. 75-91-2).

In some embodiments, the method comprises a phase of diversifying the population of vector by mutagenesis, in which the cells are incubated under conditions suitable for mutagenesis of the gene to be evolved in the absence of stringent selection for vectors having acquired a gain-of-function mutation in the gene to be evolved. In some embodiments, the method comprises a phase of stringent selection for a mutated replication product of the viral vector encoding an evolved gene.

One aspect of the PACE directed evolution methods provided herein is the mutation of the initially provided vectors encoding a gene of interest. In some embodiments, the host cells within the flow of cells in which the vector replicates comprise a mutagenesis expression construct as provided herein and are incubated under conditions that increase the natural (or basal) mutation rate. In embodiments, where the mutagenesis expression construct comprises an inducible promoter (e.g., an arabinose-inducible promoter), this may be achieved by contacting the host cells with a compound activating the inducible promoter (e.g., arabinose), in an amount sufficient to increase the mutation rate in the host cells to a desired level, or to increase the activity of the inducible promoter from a basal level to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of its maximum activity, or to fully induce the activity of the inducible promoter.

In some embodiments, the cells are not exposed to an external mutagen (e.g., a chemical mutagen, UV light, or ionizing radiation) during the PACE processes disclosed herein. In other embodiments, however, the host cells are exposed to an external mutagen in order to further increase the mutation rate in the cells. Typically, the concentration of the mutagen will be chosen to maximize the mutation rate while not being toxic to the host cells during the retention time in the lagoon. Ideally, a mutagen is used at a concentration or level of exposure that induces a desired mutation rate in a given host cell or viral vector population, but is not significantly toxic to the host cells used within the average time frame a host cell is exposed to the mutagen or the time a host cell is present in the host cell flow before being replaced by a fresh host cell. In some embodiments, the mutagen is ionizing radiation, ultraviolet radiation, base analogs, deaminating agents (e.g., nitrous acid), intercalating agents (e.g., ethidium bromide), alkylating agents (e.g., ethylnitrosourea), transposons, bromine, azide salts, psoralen, benzene,3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) (CAS no. 77439-76-0), O,O-dimethyl-S-(phthalimidomethyl)phosphorodithioate (phos-met) (CAS no. 732-11-6), formaldehyde (CAS no. 50-00-0), 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) (CAS no. 3688-53-7), glyoxal (CAS no. 107-22-2), 6-mercaptopurine (CAS no. 50-44-2), N-(trichloromethylthio)-4-cyclohexane-1,2-dicarboximide (captan) (CAS no. 133-06-2), 2-aminopurine (CAS no. 452-06-2), methyl methane sulfonate (MMS) (CAS No. 66-27-3), 4-nitroquinoline 1-oxide (4-NQO) (CAS No. 56-57-5), N4-Aminocytidine (CAS no. 57294-74-3), sodium azide (CAS no. 26628-22-8), N-ethyl-N-nitrosourea (ENU) (CAS no. 759-73-9), N-methyl-N-nitrosourea (MNU) (CAS no. 820-60-0), 5-azacytidine (CAS no. 320-67-2), cumene hydroperoxide (CHP) (CAS no. 80-15-9), ethyl methanesulfonate (EMS) (CAS no. 62-50-0), N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) (CAS no. 4245-77-6), N-methyl-N-nitro-N-nitrosoguanidine (MNNG) (CAS no. 70-25-7), 5-diazouracil (CAS no. 2435-76-9), or t-butyl hydroperoxide (BHP) (CAS no. 75-91-2). Additional suitable mutagens will be known to those of skill in the art, and the disclosure is not limited in this respect.

In some embodiments, the mutation rate of the host cells is increased by contacting the cells with a mutagenesis expression construct as provided herein. In some embodiments, the host cells are contacted with a mutagenesis plasmid. In some embodiments, the mutagenesis plasmid comprises a gene expression cassette encoding a mutagenesis-promoting gene product. In some embodiments, the mutagenesis plasmid comprises a gene expression cassette encoding umuC (a component of E. coli translesion synthesis polymerase V, e.g., as set forth in GenBank M10107.1), dam (deoxyadenosine methylase, e.g., as set forth in GenBank J01600.1), or seqA (a hemimethylated-GATC binding domain, e.g., as set forth in GenBank U07651.1), or any combination thereof. In some embodiments, the mutagenesis plasmid further comprises a nucleic acid encoding UmuD′, and/or RecA. In some embodiments, the mutagenesis plasmid comprises a gene expression cassette encoding any combination of genes or gene products provided herein, e.g., as provided in Table 2 or FIG. 7.

In some embodiments, the mutagenesis expression construct comprises an inducible promoter driving expression of at least one mutagenesis-inducing gene or gene product. Suitable inducible promoters are well known to those of skill in the art and include, for example, arabinose-inducible promoters, tetracycline or doxycyclin-inducible promoters, and tamoxifen-inducible promoters. In some embodiments, the host cell population is contacted with an inducer of the inducible promoter in an amount sufficient to effect an increased rate of mutagenesis. For example, in some embodiments, a bacterial host cell population is provided in which the host cells comprise a mutagenesis plasmid in which an expression cassette is controlled by an arabinose-inducible promoter. In some such embodiments, the population of host cells is contacted with the inducer, for example, arabinose, in an amount sufficient to induce a measurably-increased rate of mutation.

The use of an inducible mutagenesis plasmid allows one to generate a population of fresh, uninfected host cells in the absence of the inducer, thus avoiding an increased rate of mutation in the fresh host cells before they are introduced into the population of cells contacted with the viral vector. Once introduced into this population, however, these cells can then be induced to support an increased rate of mutation, which is particularly useful in some embodiments of continuous evolution. For example, in some embodiments, the host cell comprises a mutagenesis plasmid as described herein, which includes an arabinose-inducible promoter driving expression of umuC, dam, and seqA from a pBAD promoter (see, e.g., Khlebnikov A, Skaug T, Keasling J D. Modulation of gene expression from the arabinose-inducible araBAD promoter. J Ind Microbiol Biotechnol. 2002 Jul.; 29(1):34-7; incorporated herein by reference for disclosure of a pBAD promoter). In some embodiments, the fresh host cells are not exposed to arabinose, which activates expression of the above-identified genes and, thus, increases the rate of mutations in the arabinose-exposed cells, until the host cells reach the lagoon in which the population of selection phage replicates. Accordingly, in some embodiments, the mutation rate in the host cells is normal until they become part of the host cell population in the lagoon, where they are exposed to the inducer (e.g., arabinose) and, thus, to increased mutagenesis. In some embodiments, a method of continuous evolution is provided that includes a phase of diversifying the population of viral vectors by mutagenesis, in which the cells are incubated under conditions suitable for mutagenesis of the viral vector in the absence of stringent selection for the mutated replication product of the viral vector encoding the evolved protein. This is particularly useful in embodiments in which a desired function to be evolved is not merely an increase in an already present function, but the acquisition of a function not present in the gene to be evolved at the outset of the evolution procedure, such as, for example, recognition of a target promoter by a transcription factor showing no binding activity towards the target promoter in the original version of the transcription factor. A step of diversifying the pool of mutated versions of the gene of interest within the population of viral vectors, for example, of phage, allows for an increase in the chance to find a mutation that conveys the desired function, e.g., new substrate specificity in a transcription factor or enzyme.

In some embodiments, the host cell population is contacted with an agent inducing the expression of an inducible mutagenesis expression construct, such as, for example, arabinose, continuously during a PACE experiment. In other embodiments, the host cell population is contacted with the inducing agent intermittently, creating phases of increased mutagenesis, and accordingly, of increased viral vector diversification. For example, in some embodiments, the host cells are exposed to a concentration of inducing agent sufficient to generate an increased rate of mutagenesis in the gene of interest for about 10%, about 20%, about 30%, about 40%, about 50%, or about 75% of the time. In some embodiments, intermittent exposure to the encoded mutagenesis-increasing gene products can be achieved by using inducible promoters and adding or withdrawing the inducing agent from the host cell culture media during the PACE experiment.

In some embodiments of the provided methods, (1) the host cells are amenable to transfer of the vector encoding the gene to be evolved; (2) the vector allows for expression of the gene of interest in the host cell, can be replicated by the host cell, and the replicated vector can transfer into a second host cell; and (3) the host cell expresses a gene product encoded by the at least one gene for the generation of infectious phage particles (a) in response to the activity of the gene of interest, and the level of gene product expression depends on the activity of the gene of interest. The methods of directed evolution provided herein typically comprise (b) incubating the population of host cells under conditions allowing for mutation of the gene of interest, which may include induction of an inducible promoter driving expression of a nucleic acid sequence encoding one or more mutagenic gene products as provided herein, and the transfer of the vector comprising the gene of interest from host cell to host cell. The host cells are removed from the host cell population at a certain rate, e.g., at a rate that results in an average time a host cell remains in the cell population that is shorter than the average time a host cell requires to divide, but long enough for the completion of a life cycle (uptake, replication, and transfer to another host cell) of the vector. The population of host cells is replenished with fresh host cells that do not harbor the vector. In some embodiments, the rate of replenishment with fresh cells substantially matches the rate of removal of cells from the cell population, resulting in a substantially constant cell number or cell density within the cell population. The methods of directed evolution provided herein typically also comprise (c) isolating a replicated vector from the host cell population in (b), wherein the replicated vector comprises a mutated version of the gene of interest.

In some embodiments, a gene of interest is transferred from host cell to host cell in a manner dependent on the activity of the gene of interest. In some embodiments, the transfer vector is a virus infecting and replicating in the host cells, for example, a bacteriophage or a retroviral vector. In some embodiments, the viral vector is a phage vector infecting bacterial host cells. In some embodiments, the transfer vector is a retroviral vector, for example, a lentiviral vector or a vesicular stomatitis virus vector, infecting human or mouse cells. In some embodiments, the transfer vector is a conjugative plasmid transferred from a donor bacterial cell to a recipient bacterial cell.

In some embodiments, the nucleic acid vector comprising the gene of interest is a phage, a viral vector, or naked DNA (e.g., a mobilization plasmid). In some embodiments, transfer of the gene of interest from cell to cell is via infection, transfect ion, transduction, conjugation, or uptake of naked DNA, and efficiency of cell-to-cell transfer (e.g., transfer rate) is dependent on an activity of the gene of interest or a mutated version thereof. For example, in some embodiments, the nucleic acid vector is a phage harboring the gene of interest, and the efficiency of phage transfer (via infection) is dependent on the activity of the gene of interest in that a protein for the generation of infectious phage particles (e.g., pIII for M13 phage) is expressed in the host cells only in the presence of a desired activity of the gene of interest.

Some embodiments provide a continuous evolution system, in which a population of viral vectors, e.g., M13 phage vectors, comprising a gene of interest to be evolved replicates in a flow of host cells that comprise a mutagenesis expression construct provided herein, e.g., a flow through a lagoon, wherein the viral vectors are deficient in a gene encoding a protein that is essential for the generation of infectious viral particles, and wherein that gene is in the host cell under the control of a conditional promoter the activity of which depends on the activity of the gene of interest. Suitable methods, vectors, and reagents for linking the activity of a gene of interest to be evolved to expression of a gene encoding a protein that is essential for the generation of infectious viral particles are described, for example, in International PCT Application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. application, U.S. Ser. No. 13/922,812, filed Jun. 20, 2013; and U.S. application, U.S. Ser. No. 62/067,194, filed Oct. 22, 2014, the entire contents of each of which are incorporated herein by reference

Viral vectors, in which the gene of interest has not acquired a mutation conferring the desired function, will not activate the conditional promoter, or only achieve minimal activation, while any mutation in the gene of interest that confers the desired mutation will result in activation of the conditional promoter. Since the conditional promoter controls an essential protein for the viral life cycle, activation of this promoter directly corresponds to an advantage in viral spread and replication for those vectors that have acquired an advantageous mutation.

In some embodiments, the viral vector comprising the gene of interest is a phage In some embodiments, the phage is a filamentous phage. In some embodiments, the phage is an M13 phage. M13 phages are well known to those in the art and the biology of M13 phages has extensively been studied. A schematic representation of the wild-type M13 genome is provided in FIG. 16. Wild type M13 phage particles comprise a circular, single-stranded genome of approximately 6.4kb. The wild-type genome includes ten genes, gI-gX, which, in turn, encode the ten M13 proteins, pI-pX, respectively, gVIII encodes pVIII, also often referred to as the major structural protein of the phage particles, while gIII encodes pIII, also referred to as the minor coat protein, which is required for infectivity of M13 phage particles.

The M13 life cycle includes attachment of the phage to the sex pilus of a suitable bacterial host cell via the pIII protein and insertion of the phage genome into the host cell. The circular, single-stranded phage genome is then converted to a circular, double-stranded DNA, also termed the replicative form (RF), from which phage gene transcription is initiated. The wild type M13 genome comprises nine promoters and two transcriptional terminators as well as an origin of replication. This series of promoters provides a gradient of transcription such that the genes nearest the two transcriptional terminators (gVIII and IV) are transcribed at the highest levels. In wild-type M13 phage, transcription of all 10 genes proceeds in same direction. One of the phage-encode proteins, pII, initiates the generation of linear, single-stranded phage genomes in the host cells, which are subsequently circularized, and bound and stabilized by pV. The circularized, single-stranded M13 genomes are then bound by pVIII, while pV is stripped off the genome, which initiates the packaging process. At the end of the packaging process, multiple copies of pIII are attached to wild-type M13 particles, thus generating infectious phage ready to infect another host cell and concluding the life cycle.

The M13 phage genome can be manipulated, for example, by deleting one or more of the wild type genes, and/or inserting a heterologous nucleic acid construct into the genome. M13 does not have stringent genome size restrictions, and insertions of up to 42 kb have been reported. This allows M13 phage vectors to be used in continuous evolution experiments to evolve genes of interest without imposing a limitation on the length of the gene to be involved.

The M13 phage has been well characterized and the genomic sequence of M13 has been reported. Representative M13 genomic sequences can be retrieved from public databases, and an exemplary sequence is provided in entry V00604 of the National Center for Biotechnology Information (NCBI) database (www.ncbi.nlm.nih.gov):

Phage M13 genome: >gi|56713234|emb|V00604.2| Phage M13 genome (SEQ ID NO: 1) AACGCTACTACTATTAGTAGAATTGATGCCACCTTTTCAGCTCGCGCCCCAAATGAAAATAT AGCTAAACAGGTTATTGACCATTTGCGAAATGTATCTAATGGTCAAACTAAATCTACTCGTT CGCAGAATTGGGAATCAACTGTTACATGGAATGAAACTTCCAGACACCGTACTTTAGTTGCA TATTTAAAACATGTTGAGCTACAGCACCAGATTCAGCAATTAAGCTCTAAGCCATCCGCAAA AATGACCTCTTATCAAAAGGAGCAATTAAAGGTACTCTCTAATCCTGACCTGTTGGAGTTTG CTTCCGGTCTGGTTCGCTTTGAAGCTCGAATTAAAACGCGATATTTGAAGTCTTTCGGGCTT CCTCTTAATCTTTTTGATGCAATCCGCTTTGCTTCTGACTATAATAGTGAGGGTAAAGACCT GATTTTTGATTTATGGTCATTCTCGTTTTCTGAACTGTTTAAAGCATTTGAGGGGGATTCAA TGAATATTTATGACGATTCCGCAGTATTGGACGCTATCCAGTCTAAACATTTTACTATTACC CCCTCTGGCAAAACTTCTTTTGCAAAAGCCTCTCGCTATTTTGGTTTTTATCGTCGTCTGGT AAACGAGGGTTATGATAGTGTTGCTCTTACTATGCCTCGTAATTCCTTTTGGCGTTATGTAT CTGCATTAGTTGAATGTGGTATTCCTAAATCTCAACTGATGAATCTTTCTACCTGTAATAAT GTTGTTCCGTTAGTTCGTTTTATTAACGTAGATTTTTCTTCCCAACGTCCTGACTGGTATAA TGAGCCAGTTCTTAAAATCGCATAAGGTAATTCACAATGATTAAAGTTGAAATTAAACCATC TCAAGCCCAATTTACTACTCGTTCTGGTGTTTCTCGTCAGGGCAAGCCTTATTCACTGAATG AGCAGCTTTGTTACGTTGATTTGGGTAATGAATATCCGGTTCTTGTCAAGATTACTCTTGAT GAAGGTCAGCCAGCCTATGCGCCTGGTCTGTACACCGTTCATCTGTCCTCTTTCAAAGTTGG TCAGTTCGGTTCCCTTATGATTGACCGTCTGCGCCTCGTTCCGGCTAAGTAACATGGAGCAG GTCGCGGATTTCGACACAATTTATCAGGCGATGATACAAATCTCCGTTGTACTTTGTTTCGC GCTTGGTATAATCGCTGGGGGTCAAAGATGAGTGTTTTAGTGTATTCTTTCGCCTCTTTCGT TTTAGGTTGGTGCCTTCGTAGTGGCATTACGTATTTTACCCGTTTAATGGAAACTTCCTCAT GAAAAAGTCTTTAGTCCTCAAAGCCTCTGTAGCCGTTGCTACCCTCGTTCCGATGCTGTCTT TCGCTGCTGAGGGTGACGATCCCGCAAAAGCGGCCTTTAACTCCCTGCAAGCCTCAGCGACC GAATATATCGGTTATGCGTGGGCGATGGTTGTTGTCATTGTCGGCGCAACTATCGGTATCAA GCTGTTTAAGAAATTCACCTCGAAAGCAAGCTGATAAACCGATACAATTAAAGGCTCCTTTT GGAGCCTTTTTTTTTGGAGATTTTCAACATGAAAAAATTATTATTCGCAATTCCTTTAGTTG TTCCTTTCTATTCTCACTCCGCTGAAACTGTTGAAAGTTGTTTAGCAAAACCCCATACAGAA AATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGG TTGTCTGTGGAATGCTACAGGCGTTGTAGTTTGTACTGGTGACGAAACTCAGTGTTACGGTA CATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGT TCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTAT TCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACC CCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAAT AATAGGTTCCGAAATAGGCAGGGGGCATTAACTGTTTATACGGGCACTGTTACTCAAGGCAC TGACCCCGTTAAAACTTATTACCAGTACACTCCTGTATCATCAAAAGCCATGTATGACGCTT ACTGGAACGGTAAATTCAGAGACTGCGCTTTCCATTCTGGCTTTAATGAGGATCCATTCGTT TGTGAATATCAAGGCCAATCGTCTGACCTGCCTCAACCTCCTGTCAATGCTGGCGGCGGCTC TGGTGGTGGTTCTGGTGGCGGCTCTGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTG GCGGCTCTGAGGGAGGCGGTTCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAAG ATGGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGCGCTACAGTCTGA CGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGTTTCATTG GTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTCTAATTCC CAAATGGCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTTCCGTCAATATTT ACCTTCCCTCCCTCAATCGGTTGAATGTCGCCCTTTTGTCTTTAGCGCTGGTAAACCATATG AATTTTCTATTGATTGTGACAAAATAAACTTATTCCGTGGTGTCTTTGCGTTTCTTTTATAT GTTGCCACCTTTATGTATGTATTTTCTACGTTTGCTAACATACTGCGTAATAAGGAGTCTTA ATCATGCCAGTTCTTTTGGGTATTCCGTTATTATTGCGTTTCCTCGGTTTCCTTCTGGTAAC TTTGTTCGGCTATCTGCTTACTTTTCTTAAAAAGGGCTTCGGTAAGATAGCTATTGCTATTT CATTGTTTCTTGCTCTTATTATTGGGCTTAACTCAATTCTTGTGGGTTATCTCTCTGATATT AGCGCTCAATTACCCTCTGACTTTGTTCAGGGTGTTCAGTTAATTCTCCCGTCTAATGCGCT TCCCTGTTTTTATGTTATTCTCTCTGTAAAGGCTGCTATTTTCATTTTTGACGTTAAACAAA AAATCGTTTCTTATTTGGATTGGGATAAATAATATGGCTGTTTATTTTGTAACTGGCAAATT AGGCTCTGGAAAGACGCTCGTTAGCGTTGGTAAGATTCAGGATAAAATTGTAGCTGGGTGCA AAATAGCAACTAATCTTGATTTAAGGCTTCAAAACCTCCCGCAAGTCGGGAGGTTCGCTAAA ACGCCTCGCGTTCTTAGAATACCGGATAAGCCTTCTATATCTGATTTGCTTGCTATTGGGCG CGGTAATGATTCCTACGATGAAAATAAAAACGGCTTGCTTGTTCTCGATGAGTGCGGTACTT GGTTTAATACCCGTTCTTGGAATGATAAGGAAAGACAGCCGATTATTGATTGGTTTCTACAT GCTCGTAAATTAGGATGGGATATTATTTTTCTTGTTCAGGACTTATCTATTGTTGATAAACA GGCGCGTTCTGCATTAGCTGAACATGTTGTTTATTGTCGTCGTCTGGACAGAATTACTTTAC CTTTTGTCGGTACTTTATATTCTCTTATTACTGGCTCGAAAATGCCTCTGCCTAAATTACAT GTTGGCGTTGTTAAATATGGCGATTCTCAATTAAGCCCTACTGTTGAGCGTTGGCTTTATAC TGGTAAGAATTTGTATAACGCATATGATACTAAACAGGCTTTTTCTAGTAATTATGATTCCG GTGTTTATTCTTATTTAACGCCTTATTTATCACACGGTCGGTATTTCAAACCATTAAATTTA GGTCAGAAGATGAAATTAACTAAAATATATTTGAAAAAGTTTTCTCGCGTTCTTTGTCTTGC GATTGGATTTGCATCAGCATTTACATATAGTTATATAACCCAACCTAAGCCGGAGGTTAAAA AGGTAGTCTCTCAGACCTATGATTTTGATAAATTCACTATTGACTCTTCTCAGCGTCTTAAT CTAAGCTATCGCTATGTTTTCAAGGATTCTAAGGGAAAATTAATTAATAGCGACGATTTACA GAAGCAAGGTTATTCACTCACATATATTGATTTATGTACTGTTTCCATTAAAAAAGGTAATT CAAATGAAATTGTTAAATGTAATTAATTTTGTTTTCTTGATGTTTGTTTCATCATCTTCTTT TGCTCAGGTAATTGAAATGAATAATTCGCCTCTGCGCGATTTTGTAACTTGGTATTCAAAGC AATCAGGCGAATCCGTTATTGTTTCTCCCGATGTAAAAGGTACTGTTACTGTATATTCATCT GACGTTAAACCTGAAAATCTACGCAATTTCTTTATTTCTGTTTTACGTGCTAATAATTTTGA TATGGTTGGTTCAATTCCTTCCATAATTCAGAAGTATAATCCAAACAATCAGGATTATATTG ATGAATTGCCATCATCTGATAATCAGGAATATGATGATAATTCCGCTCCTTCTGGTGGTTTC TTTGTTCCGCAAAATGATAATGTTACTCAAACTTTTAAAATTAATAACGTTCGGGCAAAGGA TTTAATACGAGTTGTCGAATTGTTTGTAAAGTCTAATACTTCTAAATCCTCAAATGTATTAT CTATTGACGGCTCTAATCTATTAGTTGTTAGTGCACCTAAAGATATTTTAGATAACCTTCCT CAATTCCTTTCTACTGTTGATTTGCCAACTGACCAGATATTGATTGAGGGTTTGATATTTGA GGTTCAGCAAGGTGATGCTTTAGATTTTTCATTTGCTGCTGGCTCTCAGCGTGGCACTGTTG CAGGCGGTGTTAATACTGACCGCCTCACCTCTGTTTTATCTTCTGCTGGTGGTTCGTTCGGT ATTTTTAATGGCGATGTTTTAGGGCTATCAGTTCGCGCATTAAAGACTAATAGCCATTCAAA AATATTGTCTGTGCCACGTATTCTTACGCTTTCAGGTCAGAAGGGTTCTATCTCTGTTGGCC AGAATGTCCCTTTTATTACTGGTCGTGTGACTGGTGAATCTGCCAATGTAAATAATCCATTT CAGACGATTGAGCGTCAAAATGTAGGTATTTCCATGAGCGTTTTTCCTGTTGCAATGGCTGG CGGTAATATTGTTCTGGATATTACCAGCAAGGCCGATAGTTTGAGTTCTTCTACTCAGGCAA GTGATGTTATTACTAATCAAAGAAGTATTGCTACAACGGTTAATTTGCGTGATGGACAGACT CTTTTACTCGGTGGCCTCACTGATTATAAAAACACTTCTCAAGATTCTGGCGTACCGTTCCT GTCTAAAATCCCTTTAATCGGCCTCCTGTTTAGCTCCCGCTCTGATTCCAACGAGGAAAGCA CGTTATACGTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGC GGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTT TCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGG GGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTT GGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGG AGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCG GGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCT GATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTAAATATTTG CTTATACAATCTTCCTGTTTTTGGGGCTTTTCTGATTATCAACCGGGGTACATATGATTGAC ATGCTAGTTTTACGATTACCGTTCATCGATTCTCTTGTTTGCTCCAGACTCTCAGGCAATGA CCTGATAGCCTTTGTAGACCTCTCAAAAATAGCTACCCTCTCCGGCATGAATTTATCAGCTA GAACGGTTGAATATCATATTGATGGTGATTTGACTGTCTCCGGCCTTTCTCACCCTTTTGAA TCTTTACCTACACATTACTCAGGCATTGCATTTAAAATATATGAGGGTTCTAAAAATTTTTA TCCTTGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTATTACAGGGTCATAATGTTTTTGGTA CAACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTTAATTTTGCTAATTCTTTGCCTTGC CTGTATGATTTATTGGATGTT GENE II: join (6006..6407,1..831) (SEQ ID NO: 3) translation = MIDMLVLRLPFIDSLVCSRLSGNDLIAFVDLSKIATLSGMNSLARTVEYHIDGD LTVSGLSHPFESLPTHYSGIAFKIYEGSKNFYPCVEIKASPAKVLQGHNVFGTTDLALCSEA LLLNFANSLPCLYDLLDVNATTISRIDATFSARAPNENIAKQVIDHLRNVSNGQTKSTRSQN WESTVTWNETSRHRTLVAYLKHVELQHQIQQLSSKPSAKMTSYQKEQLKVLSNPDLLEFASG LVRFEARIKTRYLKSFGLPLNLFDAIRFASDYNSQGKDLIFDLWSFSFSELFKAFEGDSMNI YDDSAVLDAIQSKHFTITPSGKTSFAKASRYFGFYRRLVNEGYDSVALTMPRNSFWRYVSAL VECGIPKSQLMNLSTCNNVVPLVRFINVDFSSQRPDWYNEPVLKIA GENE X: 496.831 (SEQ ID NO: 3) translation = MNIYDDSAVLDAIQSKHFTITPSGKTSFAKASRYFGFYRRLVNEGYDSVALTMP RNSFWRYVSALVECGIPKSQLMNLSTCNNVVPLVRFINVDFSSQRPDWYNEPVLKIA GENE V: 843..1106 (SEQ ID NO: 4) translation = MIKVEIKPSQAQFTTRSGVSRQGKPYSLNEQLCYVDLGNEYPVLKITLDEGQP AYAPGLYTVHLSSFKVGQFGSLMIDRLRLVPAK GENE VII: 1108..1209 (SEQ ID NO: 5) translation = MEQVADFDTIYQAMIQISVVLCFALGIIAGGQR GENE IX: 1206..1304 (SEQ ID NO: 6) translation = MSVLVYSFASFVLGWCLRSGITYFTRLMETSS GENE VIII: 1301..1522 (SEQ ID NO: 7) translation = MKKSLVLKASVAVATLVPMLSFAAEGDDPAKAAFNSLQASATEYIGYAWAMVVV IVGATIGIKLFKKFTSKAS GENE III: 1579..2853 (SEQ ID NO: 8) translation = MKKLLFAIPLVVPFYSHSAETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGC LWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIP GYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRPRNRQGALTVYTGTVTQGTD PVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPVNAGGGSG GGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADENALQSDA KGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLP SLPQSVECRPFVFSAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES GENE VI: 2856..3194 (SEQ ID NO: 9) translation = MPVLLGIPLLLRFLGFLLVTLFGYLLTFLKKGFGKIAIAISLFLALIIGLNSIL VGYLSDISAQLPSDFVQGVQLILPSNALPCFYVILSVKAAIFIFDVKQKIVSYLDWDK GENE I: 3196..4242 (SEQ ID NO: 10) translation = MAVYFVTGKLGSGKTLVSVGKIQDKIVAGCKIATNLDLRLQNLPQVGRFAKTPR VLRIPDKPSISDLLAIGRGNDSYDENKNGLLVLDECGTWFNTRSWNDKERQPIIDWFLHARK LGWDIIFLVQDLSIVDKQARSALAEHVVYCRRLDRITLPFVGTLYSLITGSKMPLPKLHVGV VKYGDSQLSPTVERWLYTGKNLYNAYDTKQAFSSNYDSGVYSYLTPYLSHGRYFKPLNLGQK MKLTKIYLKKFSRVLCLAIGFASAFTYSYITQPKPEVKKVVSQTYDFDKFTIDSSQRLNLSY RYVFKDSKGKLINSDDLQKQGYSLTYIDLCTVSIKKGNSNEIVKCN GENE IV: 4220..5500 (SEQ ID NO: 11) translation = MKLLNVINFVFLMFVSSSSFAQVIEMNNSPLRDFVTWYSKQSGESVIVSPDVKG TVTVYSSDVKPENLRNFFISVLRANNFDMVGSIPSIIQKYNPNNQDYIDELPSSDNQEYDDN SAPSGGFFVPQNDNVTQTFKINNVRAKDLIRVVELFVKSNTSKSSNVLSIDGSNLLVVSAPK DILDNLPQFLSTVDLPTDQILIEGLIFEVQQGDALDFSFAAGSQRGTVAGGVNTDRLTSVLS SAGGSFGIFNGDVLGLSVRALKTNSHSKILSVPRILTLSGQKGSISVGQNVPFITGRVTGES ANVNNPFQTIERQNVGISMSVFPVAMAGGNIVLDITSKAOSLSSSTQASDVITNQRSIATTV NLRDGQTLLLGGLTDYKNTSQDSGVPFLSKIPLIGLLFSSR3DSNEESTLYVLVKATIVRAL

Some embodiments of the PACE technology described herein utilize a “selection phage,” a modified phage that comprises a gene of interest to be evolved and lacks a full-length gene encoding a protein required for the generation of infectious phage particles. In some such embodiments, the selection phage serves as the vector that replicates and evolves in the flow of host cells. For example, some M13 selection phages provided herein comprise a nucleic acid sequence encoding a gene to be evolved, e.g., under the control of an M13 promoter, and lack all or part of a phage gene encoding a protein required for the generation of infectious phage particles, e.g., gI, gII, gIII, gIV, gV, gVI, gVII, gVIII, gIX, or gX, or any combination thereof. For example, some M13 selection phages provided herein comprise a nucleic acid sequence encoding a gene product, e.g., a transcript or a protein, to be evolved, e.g., under the control of an M13 promoter, and lack all or part of a gene encoding a protein required for the generation of infective phage particles, e.g., the gIII gene encoding the pII protein. An exemplary, non-limiting selection plasmid sequence, SP-MCS, comprising a multiple cloning site, into which a nucleic acid sequence encoding a gene product to be evolved can be cloned, is provided below:

(SEQ ID NO: 30) ATGATTGACATGCTAGTTTTACGATTACCGTTCATCGATTCTCTTGTTTGCTCCAGACTCTC AGGCAATGACCTGATAGCCTTTGTAGACCTCTCAAAAATAGCTACCCTCTCCGGCATGAATT TATCAGCTAGAACGGTTGAATATCATGTTGATGGTGATTTGACTGTCTCCGGCCTTTCTCAC CCTTTTGAATCTTTACCTACACATTACTCAGGCATTGCATTTAAAATATATGAGGGTTCTAA AAATTTTTATCCTTGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTATTACAGGGTCATAATG TTTTTGGTACAACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTTAATTTTGCTAATTCT TTGCCTTGCCTGTATGATTTATTGGATGTTAACGCTACTACTATTAGTAGAATTGATGCCAC CTTTTCAGCTCGCGCCCCAAATGAAAATATAGCTAAACAGGTTATTGACCATTTGCGAAATG TATCTAATGGTCAAACTAAATCTACTCGTTCGCAGAATTGGGAATCAACTGTTACATGGAAT GAAACTTCCAGACACCGTACTTTAGTTGCATATTTAAAACATGTTGAGCTACAGCACCAGAT TCAGCAATTAAGCTCTAAGCCATCCGCAAAAATGACCTCTTATCAAAAGGAGCAATTAAAGG TACTCTCTAATCCTGACCTGTTGGAGTTTGCTTCCGGGCTGGTTCGCTTTGAAGCTCGAATT AGAACGCGATATTTGAAGTCTTTCGGGCTTCCTCTTAATCTTTTTGATGCAATCCGCTTTGC TTCTGACTATAATAGTCAGGGTAAAGACCTGATTTTTGATTTATGGTCATTCTCGTTTTCTG AACTGTTTAAAGCATTTGAGGGGGATTCAATGAATATTTATGACGATTCCGCAGTATTGGAC GCTATCCAGTCTAAACATTTTACTATTACCCCCTCTGGCAAAACTTCTTTTGCAAAAGCCTC TCGCTATTTTGGTTTTTATCGTCGTCTGGTAAACGAGGGTTATGATAGTGTTGCTCTTACTA TGCCTCGTAATTCCTTTTGGCGTTATGTATCTGCATTAGTTGAATGTGGTATTCCTAAATCT CAACTGATGAATCTTTCTACCTGTAATAATGTTGTTCCGTTAGTTCGTTTTATTAACGTAGA TTTTTCTTCCCAACGTCCTGACTGGTATAATGAGCCAGTTCTTAAAATCGCATAAGGTAATT CACAATGATTAAAGTTGAAATTAAACCATCTCAAGCCCAATTTACTACTCGTTCTGGTGTTT CTCGTCAGGGCAAGCCTTATTCACTGAATGAGCAGCTTTGTTACGTTGATTTGGGTAATGAA TATCCGGTTCTTGTCAAGATTACTCTTGATGAAGGTCAGCCAGCCTATGCGCCTGGTCTGTA CACCGTTCATCTGTCCTCTTTCAAAGTTGGTCAGTTCGGTTCCCTTATGATTGACCGTCTGC GCCTCGTTCCGGCTAAGTAACATGGAGCAGGTCGCGGATTTCGACACAATTTATCAGGCGAT GATACAAATCTCCGTTGTACTTTGTTTCGCGCTTGGTATAATCGCTGGGGGTCAAAGATGAG TGTTTTAGTGTATTCTTTCGCCTCTTTCGTTTTAGGTTGGTGCCTTCGTAGTGGCATTACGT ATTTTACCCGTTTAATGGAAACTTCCTCATGAAAAAGTCTTTAGTCCTCAAAGCCTCTGTAG CCGTTGCTACCCTCGTTCCGATGCTGTCTTTCGCTGCTGAGGGTGACGATCCCGCAAAAGCG GCCTTTAACTCCCTGCAAGCCTCAGCGACCGAATATATCGGTTATGCGTGGGCGATGGTTGT TGTCATTGTCGGCGCAACTATCGGTATCAAGCTGTTTAAGAAATTCACCTCGAAAGCAAGCT GATAAACCGATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTCGCGCCAGAAGGAGACCA AGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCTGGAGA TTTTCAACATGCTCCCTCAATCGGTTGAATGTCGCCCTTTTGTCTTTAGCGCTGGTAAACCA TATGAATTTTCTATTGATTGTGACAAAATGAACTTATTCCGTGGTGTCTTTGCGTTTCTTTT ATATGTTGCCACCTTTATGTATGTATTTTCTACGTTTGCTAACATACTGCGTAATAAGGAGT CTTAATCATGCCAGTTCTTTTGGGTATTCCGTTATTATTGCGTTTCCTCGGTTTCCTTCTGG TAACTTTGTTCGGCTATCTGCTTACTTTTCTTAAAAAGGGCTTCGGTAAGATAGCTATTGCT ATTTCATTGTTTCTTGCTCTTATTATTGGGCTTAACTCAATTCTTGTGGGTTATCTCTCTGA TATTAGCGCTCAATTACCCTCTGACTTTGTTCAGGGTGTTCAGTTAATTCTCCCGTCTAATG CGCTTCCCTGTTTTTATGTTATTCTCTCTGTAAAGGCTGCTATTTTCATTTTTGACGTTAAA CAAAAAATCGTTTCTTATTTGGATTGGGATAAATAATATGGCTGTTTATTTTGTAACTGGCA AATTAGGCTCTGGAAAGACGCTCGTTAGCGTTGGTAAGATTCAGGATAAAATTGTAGCTGGG TGCAAAATAGCAACTAATCTTGATTTAAGGCTTCAAAACCTCCCGCAAGTCGGGAGGTTCGC TAAAACGCCTCGCGTTCTTAGAATACCGGATAAGCCTTCTATATCTGATTTGCTTGCTATTG GGCGCGGTAATGATTCCTACGATGAAAATAAAAACGGCTTGCTTGTTCTCGATGAGTGCGGT ACTTGGTTTAATACCCGTTCTTGGAATGATAAGGAAAGACAGCCGATTATTGATTGGTTTCT ACATGCTCGTAAATTAGGATGGGATATTATTTTTCTTGTTCAGGACTTATCTATTGTTGATA AACAGGCGCGTTCTGCATTAGCTGAACATGTTGTTTATTGTCGTCGTCTGGACAGAATTACT TTACCTTTTGTCGGTACTTTATATTCTCTTATTACTGGCTCGAAAATGCCTCTGCCTAAATT ACATGTTGGCGTTGTTAAATATGGCGATTCTCAATTAAGCCCTACTGTTGAGCGTTGGCTTT ATACTGGTAAGAATTTGTATAACGCATATGATACTAAACAGGCTTTTTCTAGTAATTATGAT TCCGGTGTTTATTCTTATTTAACGCCTTATTTATCACACGGTCGGTATTTCAAACCATTAAA TTTAGGTCAGAAGATGAAATTAACTAAAATATATTTGAAAAAGTTTTCTCGCGTTCTTTGTC TTGCGATTGGATTTGCATCAGCATTTACATATAGTTATATAACCCAACCTAAGCCGGAGGTT AAAAAGGTAGTCTCTCAGACCTATGATTTTGATAAATTCACTATTGACTCTTCTCAGCGTCT TAATCTAAGCTATCGCTATGTTTTCAAGGATTCTAAGGGAAAATTAATTAATAGCGACGATT TACAGAAGCAAGGTTATTCACTCACATATATTGATTTATGTACTGTTTCCATTAAAAAAGGT AATTCAAATGAAATTGTTAAATGTAATTAATTTTGTTTTCTTGATGTTTGTTTCATCATCTT CTTTTGCTCAGGTAATTGAAATGAATAATTCGCCTCTGCGCGATTTTGTAACTTGGTATTCA AAGCAATCAGGCGAATCCGTTATTGTTTCTCCCGATGTAAAAGGTACTGTTACTGTATATTC ATCTGACGTTAAACCTGAAAATCTACGCAATTTCTTTATTTCTGTTTTACGTGCAAGTAATT TTGATATGGTTGGTTCTAACCCTTCCATTATTCAGAAGTATAATCCAAACAATCAGGATTAT ATTGATGAATTGCCATCATCTGATAATCAGGAATATGATGATAATTCCGCTCCTTCTGGTGG TTTCTTTGTTCCGCAAAATGATAATGTTACTCAAACTTTTAAAATTAATAACGTTCGGGCAA AGGATTTAATACGAGTTGTCGAATTGTTTGTAAAGTCTAATACTTCTAAATCCTCAAATGTA TTATCTATTGACGGCTCTAATCTATTAGTTGTTAGTGCACCTAAAGATATTTTAGATAACCT TCCTCAATTCCTTTCTACTGTTGATTTGCCAACTGACCAGATATTGATTGAGGGTTTGATAT TTGAGGTTCAGCAAGGTGATGCTTTAGATTTTTCATTTGCTGCTGGCTCTCAGCGTGGCACT GTTGCAGGCGGTGTTAATACTGACCGCCTCACCTCTGTTTTATCTTCTGCTGGTGGTTCGTT CGGTATTTTTAATGGCGATGTTTTAGGGCTATCAGTTCGCGCATTAAAGACTAATAGCCATT CAAAAATATTGTCTGTGCCACGTATTCTTACGCTTTCAGGTCAGAAGGGTTCTATCTTTGTT GGCCAGAATGTCCCTTTTATTACTGGTCGTGTGACTGGTGAATCTGCCAATGTAAATAATCC ATTTCAGACGATTGAGCGTCAAAATGTAGGTATTTCCATGAGCGTTTTTCCTGTTGCAATGG CTGGCGGTAATATTGTTCTGGATATTACCAGCAAGGCCGATAGTTTGAGTTCTTCTACTCAG GCAAGTGATGTTATTACTAATCAAAGAAGTACTGCTACAACGGTTAATTTGCGTGATGGACA GACTCTTTTACTCGGTGGCCTCACTGATTATAAAAACACTTCTCAGGATTCTGGCGTACCGT TCCTGTCTAAAATCCCTTTAATCGGCCTCCTGTTTAGCTCCCGCTCTGATTCTAACGAGGAA AGCACGTTATACGTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCG CGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCT CCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAA TCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTG ATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACG TTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTAT CTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATG AGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTAAATA TTTGCTTATACAATCTTCCTGTTTTTGGGGCTTTTCTTATTATCAACCGGGGTACAT

In some embodiments, the directed evolution methods provided herein comprise an initial or intermittent phase of diversifying the population of vectors by mutagenesis, in which the cells are incubated under conditions suitable for mutagenesis of the gene of interest in the absence of stringent selection or in the absence of any selection for evolved variants of the gene to be evolved that have acquired a desired activity. Such low-stringency selection or no selection periods may be achieved by supporting expression of the gene for the generation of infectious phage particles in the absence of desired activity of the gene to be evolved, for example, by providing an inducible expression construct comprising a gene encoding the respective phage packaging protein under the control of an inducible promoter and incubating under conditions that induce expression of the promoter, e.g., in the presence of the inducing agent. Suitable inducible promoters and inducible expression systems are described herein and in International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; and U.S. application, U.S. Ser. No. 13/922,812, filed Jun. 20, 2013, the entire contents of each of which are incorporated herein by reference. Additional suitable promoters and inducible gene expression systems will be apparent to those of skill in the art based on the instant disclosure. In some embodiments, the method comprises a phase of stringent selection for a mutated version of the gene to be evolved. If an inducible expression system is used to relieve selective pressure, the stringency of selection can be increased by removing the inducing agent from the population of cells in the lagoon, thus turning expression from the inducible promoter off, so that any expression of the gene required for the generation of infectious phage particles must come from the expression system that depends on the activity of the gene product to be evolved, e.g., the conditional promoter-controlled version of the gene required for the generation of infectious phage particles comprised in an accessory plasmid.

In some embodiments, the link between desired gene product activity and selective advantage for an encoding phage is provided by an expression system in which at least one gene for the generation of infectious phage particles is expressed in response to the desired activity of the gene to be evolved as described in more detail elsewhere herein. In some embodiments, the at least one gene for the generation of infectious phage particles to another host cell is a gene required for the production of infectious phage particles. In some embodiments, the vector is M13 phage, and the at least one gene for the generation of infectious phage particles comprises a full-length M13 pIII gene. In some embodiments, the host cells comprise an accessory plasmid comprising an expression construct encoding the at least one gene for the generation of infectious phage particles, e.g., the full-length pIII protein, under the control of a conditional promoter that is activated by a desired function of the gene to be evolved.

In some embodiments, the conditional promoter of the accessory plasmid is a promoter, the transcriptional activity of which can be regulated over a wide range, for example, over 2, 3, 4, 5, 6, 7, 8, 9, or 10 orders of magnitude by the activating function, for example, the function of a gene of interest. In some embodiments, the conditional promoter has a basal activity that allows for baseline packaging of viral vectors even in the absence of the desired activity of the gene to be evolved or in the presence of only minimal desired activity of the gene to be evolved. This allows for starting a continuous evolution process with a viral vector population comprising versions of the gene of interest that only show minimal activation of the conditional promoter, e.g., of a wild-type version of the gene of interest. In the process of continuous evolution, any mutation in the gene of interest that increases activity of the conditional promoter directly translates into higher expression levels of the gene required for the generation of infectious viral particles in the host cells harboring the vector comprising such a mutation, and, thus, into a competitive advantage over other viral vectors carrying minimally active or loss-of-function versions of the gene of interest.

One function of the accessory plasmid is to provide a gene for the generation of infectious phage particles under the control of a conditional promoter the activity of which depends on a function of the gene of interest. Accordingly, the accessory plasmid provides a selection mechanism that favors desirable mutations over inconsequential mutations or mutations that are detrimental to the desired function. The stringency of selective pressure imposed by the accessory plasmid in a continuous evolution procedure as provided herein can be modulated. For example, an accessory plasmid may be used at different copy numbers per cell, may comprise a conditional promoter having a base line transcription rate (“leakiness”) that prevents washout of unmutated sequences from the lagoon while still providing a selective advantage to desirable mutations. In some embodiments, an accessory plasmid comprising an expression cassette in which the gene for the generation of infectious phage particles is under the control of an inducible promoter that can be activated by a chemical compound (e.g., a tet-on promoter), allowing for titration of the expression of the gene for the generation of infectious phage particles during a continuous evolution experiment.

In some embodiments, the use of low copy number accessory plasmids results in an elevated stringency of selection for versions of the gene of interest that activate the conditional promoter on the accessory plasmid, while the use of high copy number accessory plasmids results in a lower stringency of selection. The copy number of an accessory plasmid will depend to a large part on the origin of replication employed. Those of skill in the art will be able to determine suitable origins of replication in order to achieve a desired copy number. The following table lists some non-limiting examples of vectors of different copy numbers and with different origins of replication:

Origin of Copy Plasmids Replication number Class pUC vectors pMB1* 500-700 high copy pBluescript ® vectors ColE1 300-500 high copy pGEM ® vectors pMB1* 300-400 high copy pTZ vectors pMB1* >1000 high copy pBR322 and derivatives pMB1* 15-20 low copy pACYC and derivatives p15A 10-12 low copy pSC101 and derivatives pSC101 ~5 very low copy *The pMB1 origin of replication is closely related to that of ColE1 and falls in the same incompatibility group. The high-copy plasmids listed here contain mutated versions of this origin.

It should be understood that one function of the accessory plasmid, namely to provide a gene for the generation of infectious phage particles under the control of a conditional promoter, the activity of which depends on a function of the gene of interest, can be conferred to a host cell in alternative ways. Such alternatives include, but are not limited to, permanent insertion of a gene construct comprising the conditional promoter and the respective gene into the genome of the host cell, or introducing it into the host cell using a different vector, for example, a phagemid, a cosmid, a phage, a virus, or an artificial chromosome. Additional ways to confer accessory plasmid function to host cells will be evident to those of skill in the art, and the invention is not limited in this respect.

The sequences of two exemplary, non-limiting accessory plasmids, AP-MCS-A, and AP-MCS-P, respectively, are provided below:

AP-MCS-A: (SEQ ID NO: 31) GGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAG CGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGC CGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTA GGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTA TCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAAC GTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCA AATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTACTCTGCT AGCAAGTAAGGCCGACAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCG AGCTCGAATTCCCTTTTTTTTTGGAGATTTTCAACGTGAAAAAATTATTATTCGCAATTCCT TTAGTTGTTCCTTTCTATTCTCACTCCGCTGAAACTGTTGAAAGTTGTTTAGCAAAACCCCA TACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACT ATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTAGTTTGTACTGGTGACGAAACTCAGTGT TACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGG TGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATA CACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAG CAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTT TCAGAATAATAGGTTCCGAAATAGGCAGGGGGCATTAACTGTTTATACGGGCACTGTTACTC AAGGCACTGACCCCGTTAAAACTTATTACCAGTACACTCCTGTATCATCAAAAGCCATGTAT GACGCTTACTGGAACGGTAAATTCAGAGACTGCGCTTTCCATTCTGGCTTTAATGAGGATCC ATTCGTTTGTGAATATCAAGGCCAATCGTCTGACCTGCCTCAACCTCCTGTCAATGCTGGCG GCGGCTCTGGTGGTGGTTCTGGTGGCGGCTCTGAGGGTGGTGGCTCTGAGGGTGGCGGTTCT GAGGGTGGCGGCTCTGAGGGAGGCGGTTCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTA TGAAAAGATGGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGCGCTAC AGTCTGACGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGT TTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTC TAATTCCCAAATGGCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTTCCGTC AATATTTACCTTCCCTCCCTCAATCGGTTGAATGTCGCCCTTTTGTCTTTGGCGCTGGTAAA CCATATGAATTTTCTATTGATTGTGACAAAATAAACTTATTCCGTGGTGTCTTTGCGTTTCT TTTATATGTTGCCACCTTTATGTATGTATTTTCTACGTTTGCTAACATACTGCGTAATAAGG AGTCTTAATCATGCCAGTTCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTT TTTTGCCTTGTCGGCCTTACTTGCTAAATACATTCAAATATGTATCCGCTCATGAGACAATA ACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTG TCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTG GTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCT CAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTT TTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCT TACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTG CGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAAC ATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAA CGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTG GCGAACTACTTACTCTAGCTTCCCGGCAACAATTGATAGACTGGATGGAGGCGGATAAAGTT GCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC CGGTGAGCGTGGCTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTA TCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCT GAGATAGGTGCCTCACTGATTAAGCATTGGTAAGAACCTCAGATCCTTCCGTGATGGTAACT TCACTAGTTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT AACGTGAGTTTTCGTTCCACTGAGCGTCAGAGAACCTCAGATCCTTCCGTATTTAGCCAGTA TGTTCTCTAGTGTGGTTCGTTGTTTTTGCGTGAGCCATGAGAACGAACCATTGAGATCATGC TTACTTTGCATGTCACTCAAAAATTTTGCCTCAAAACTGGTGAGCTGAATTTTTGCAGTTAA AGCATCGTGTAGTGTTTTTCTTAGTCCGTTACGTAGGTAGGAATCTGATGTAATGGTTGTTG GTATTTTGTCACCATTCATTTTTATCTGGTTGTTCTCAAGTTCGGTTACGAGATCCATTTGT CTATCTAGTTCAACTTGGAAAATCAACGTATCAGTCGGGCGGCCTCGCTTATCAACCACCAA TTTCATATTGCTGTAAGTGTTTAAATCTTTACTTATTGGTTTCAAAACCCATTGGTTAAGCC TTTTAAACTCATGGTAGTTATTTTCAAGCATTAACATGAACTTAAATTCATCAAGGCTAATC TCTATATTTGCCTTGTGAGTTTTCTTTTGTGTTAGTTCTTTTAATAACCACTCATAAATCCT CATAGAGTATTTGTTTTCAAAAGACTTAACATGTTCCAGATTATATTTTATGAATTTTTTTA ACTGGAAAAGATAAGGCAATATCTCTTCACTAAAAACTAATTCTAATTTTTCGCTTGAGAAC TTGGCATAGTTTGTCCACTGGAAAATCTCAAAGCCTTTAACCAAAGGATTCCTGATTTCCAC AGTTCTCGTCATCAGCTCTCTGGTTGCTTTAGCTAATACACCATAAGCATTTTCCCTACTGA TGTTCATCATCTGAGCGTATTGGTTATAAGTGAACGATACCGTCCGTTCTTTCCTTGTAGGG TTTTCAATCGTGGGGTTGAGTAGTGCCACACAGCATAAAATTAGCTTGGTTTCATGCTCCGT TAAGTCATAGCGACTAATCGCTAGTTCATTTGCTTTGAAAACAACTAATTCAGACATACATC TCAATTGGTCTAGGTGATTTTAATCACTATACCAATTGAGATGGGCTAGTCAATGATAATTA CTAGTCCTTTTCCTTTGAGTTGTGGGTATCTGTAAATTCTGCTAGACCTTTGCTGGAAAACT TGTAAATTCTGCTAGACCCTCTGTAAATTCCGCTAGACCTTTGTGTGTTTTTTTTGTTTATA TTCAAGTGGTTATAATTTATAGAATAAAGAAAGAATAAAAAAAGATAAAAAGAATAGATCCC AGCCCTGTGTATAACTCACTACTTTAGTCAGTTCCGCAGTATTACAAAAGGATGTCGCAAAC GCTGTTTGCTCCTCTACAAAACAGACCTTAAAACCCTAAAGGCTTAAGTAGCACCCTCGCAA GCTCGGGCAAATCGCTGAATATTCCTTTTGTCTCCGACCATCAGGCACCTGAGTCGCTGTCT TTTTCGTGACATTCAGTTCGCTGCGCTCACGGCTCTGGCAGTGAATGGGGGTAAATGGCACT ACAGGCGCCTTTTATGGATTCATGCAAGGAAACTACCCATAATACAAGAAAAGCCCGTCACG GGCTTCTCAGGGCGTTTTATGGCGGGTCTGCTATGTGGTGCTATCTGACTTTTTGCTGTTCA GCAGTTCCTGCCCTCTGATTTTCCAGTCTGACCACTTCGGATTATCCCGTGACAGGTCATTC AGACTGGCTAATGCACCCAGTAAGGCAGCGGTATCATCAACT AP-MCS-P: (SEQ ID NO: 32) GGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAG CGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGC CGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTA GGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTA TCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAAC GTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCA AATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTACTCTGCT AGCAAGTAAGGCCGACAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCG AGCTCGAATTCCCTTTTTTTTTGGAGATTTTCAACGTGAAAAAATTATTATTCGCAATTCCT TTAGTTGTTCCTTTCTATTCTCACTCCGCTGAAACTGTTGAAAGTTGTTTAGCAAAACCCCA TACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACT ATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTAGTTTGTACTGGTGACGAAACTCAGTGT TACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGG TGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATA CACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAG CAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTT TCAGAATAATAGGTTCCGAAATAGGCAGGGGGCATTAACTGTTTATACGGGCACTGTTACTC AAGGCACTGACCCCGTTAAAACTTATTACCAGTACACTCCTGTATCATCAAAAGCCATGTAT GACGCTTACTGGAACGGTAAATTCAGAGACTGCGCTTTCCATTCTGGCTTTAATGAGGATCC ATTCGTTTGTGAATATCAAGGCCAATCGTCTGACCTGCCTCAACCTCCTGTCAATGCTGGCG GCGGCTCTGGTGGTGGTTCTGGTGGCGGCTCTGAGGGTGGTGGCTCTGAGGGTGGCGGTTCT GAGGGTGGCGGCTCTGAGGGAGGCGGTTCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTA TGAAAAGATGGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGCGCTAC AGTCTGACGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGT TTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTC TAATTCCCAAATGGCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTTCCGTC AATATTTACCTTCCCTCCCTCAATCGGTTGAATGTCGCCCTTTTGTCTTTGGCGCTGGTAAA CCATATGAATTTTCTATTGATTGTGACAAAATAAACTTATTCCGTGGTGTCTTTGCGTTTCT TTTATATGTTGCCACCTTTATGTATGTATTTTCTACGTTTGCTAACATACTGCGTAATAAGG AGTCTTAATCATGCCAGTTCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTT TTTTGCCTTGTCGGCCTTACTTGCTAAATACATTCAAATATGTATCCGCTCATGAGACAATA ACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTG TCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTG GTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCT CAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTT TTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCT TACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTG CGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAAC ATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAA CGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTG GCGAACTACTTACTCTAGCTTCCCGGCAACAATTGATAGACTGGATGGAGGCGGATAAAGTT GCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC CGGTGAGCGTGGCTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTA TCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCT GAGATAGGTGCCTCACTGATTAAGCATTGGTAAGAACCTCAGATCCTTCCGTGATGGTAACT TCACTAGTTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT AACGTGAGTTTTCGTTCCACTGAGCGTCAGAGAACCTCAGATCCTTCCGTATTTAGCCAGTA TGTTCTCTAGTGTGGTTCGTTGTTTTTGCGTGAGCCATGAGAACGAACCATTGAGATCATGC TTACTTTGCATGTCACTCAAAAATTTTGCCTCAAAACTGGTGAGCTGAATTTTTGCAGTTAA AGCATCGTGTAGTGTTTTTCTTAGTCCGTTACGTAGGTAGGAATCTGATGTAATGGTTGTTG GTATTTTGTCACCATTCATTTTTATCTGGTTGTTCTCAAGTTCGGTTACGAGATCCATTTGT CTATCTAGTTCAACTTGGAAAATCAACGTATCAGTCGGGCGGCCTCGCTTATCAACCACCAA TTTCATATTGCTGTAAGTGTTTAAATCTTTACTTATTGGTTTCAAAACCCATTGGTTAAGCC TTTTAAACTCATGGTAGTTATTTTCAAGCATTAACATGAACTTAAATTCATCAAGGCTAATC TCTATATTTGCCTTGTGAGTTTTCTTTTGTGTTAGTTCTTTTAATAACCACTCATAAATCCT CATAGAGTATTTGTTTTCAAAAGACTTAACATGTTCCAGATTATATTTTATGAATTTTTTTA ACTGGAAAAGATAAGGCAATATCTCTTCACTAAAAACTAATTCTAATTTTTCGCTTGAGAAC TTGGCATAGTTTGTCCACTGGAAAATCTCAAAGCCTTTAACCAAAGGATTCCTGATTTCCAC AGTTCTCGTCATCAGCTCTCTGGTTGCTTTAGCTAATACACCATAAGCATTTTCCCTACTGA TGTTCATCATCTGAGCGTATTGGTTATAAGTGAACGATACCGTCCGTTCTTTCCTTGTAGGG TTTTCAATCGTGGGGTTGAGTAGTGCCACACAGCATAAAATTAGCTTGGTTTCATGCTCCGT TAAGTCATAGCGACTAATCGCTAGTTCATTTGCTTTGAAAACAACTAATTCAGACATACATC TCAATTGGTCTAGGTGATTTTAATCACTATACCAATTGAGATGGGCTAGTCAATGATAATTA CTAGTCCTTTTCCTTTGAGTTGTGGGTATCTGTAAATTCTGCTAGACCTTTGCTGGAAAACT TGTAAATTCTGCTAGACCCTCTGTAAATTCCGCTAGACCTTTGTGTGTTTTTTTTGTTTATA TTCAAGTGGTTATAATTTATAGAATAAAGAAAGAATAAAAAAAGATAAAAAGAATAGATCCC AGCCCTGTGTATAACTCACTACTTTAGTCAGTTCCGCAGTATTACAAAAGGATGTCGCAAAC GCTGTTTGCTCCTCTACAAAACAGACCTTAAAACCCTAAAGGCTTAAGTAGCACCCTCGCAA GCTCGGGCAAATCGCTGAATATTCCTTTTGTCTCCGACCATCAGGCACCTGAGTCGCTGTCT TTTTCGTGACATTCAGTTCGCTGCGCTCACGGCTCTGGCAGTGAATGGGGGTAAATGGCACT ACAGGCGCCTTTTATGGATTCATGCAAGGAAACTACCCATAATACAAGAAAAGCGCGTCACG GGCTTCTCAGGGCGTTTTATGGCGGGTCTGCTATGTGGTGCTATCTGACTTTTTGCTGTTCA GCAGTTCCTGCCCTCTGATTTTCCAGTCTGACCACTTCGGATTATCCCGTGACAGGTCATTC AGACTGGCTAATGCACCCAGTAAGGCAGCGGTATCATCAACT

In some embodiments, the gene to be evolved encodes a transcription factor that can directly drive expression from a conditional promoter, resulting in a relatively straightforward linkage of activity of the gene to be evolved to phage packaging efficiency. In embodiments where the gene of interest encodes a gene product that cannot directly drive transcription from a promoter, the linkage of activity of the gene to be evolved to viral particle packaging is provided indirectly, for example, as described in International PCT application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. application, U.S. Ser. No. 13/922,812, filed Jun. 20, 2013; and U.S. application, U.S. Ser. No. 62/067,194, filed Oct. 22, 2014, the entire contents of each of which are incorporated herein by reference.

In some embodiments, the host cells comprise all phage genes except for the at least one gene for the generation of infectious phage particles in the form of a helper phage. In some embodiments, the phage genes on the helper phage include pI, pII, pIV, pV, pVI, pVII, pVIII, pIX, and/or pX. Some exemplary helper phages suitable for use in such embodiments are provided herein, and additional suitable helper phages will be apparent to the skilled artisan based on the instant disclosure. The host cell may also provide phage functions based on expression constructs other than helper phage, for example, expression constructs integrated into the host cell genome or provided on artificial chromosomes or on separate plasmids. One advantage of providing phage functions in the host cell, e.g., by using a helper phage, is that the selection phage encoding the gene of interest can be deficient in genes encoding proteins or other functions provided by the host cell and can, accordingly, carry a longer gene of interest.

In some embodiments, diversifying the vector population is achieved by providing a flow of host cells that does not select for gain-of-function mutations in the gene of interest for replication, mutagenesis, and propagation of the population of vectors. In some embodiments, the host cells are host cells that express all genes required for the generation of infectious viral particles, for example, bacterial cells that express a complete helper phage, and, thus, do not impose selective pressure on the gene of interest. In other embodiments, the host cells comprise an accessory plasmid comprising a conditional promoter with a baseline activity sufficient to support viral vector propagation even in the absence of significant gain-of-function mutations of the gene of interest. This can be achieved by using a “leaky” conditional promoter, by using a high-copy number accessory plasmid, thus amplifying baseline leakiness, and/or by using a conditional promoter on which the initial version of the gene of interest effects a low level of activity while a desired gain-of-function mutation effects a significantly higher activity.

Such methods involving host cells of varying selective stringency or varying the selection stringency in other ways as described herein allow for harnessing the power of continuous evolution methods as provided herein for the evolution of functions that are completely absent in the initial version of the gene of interest, for example, for the evolution of enzymes that bind substrates not recognized by the initial enzyme used at the outset of the respective PACE experiments at all.

In some embodiments, the PACE methods provided herein further comprises a negative selection for undesired activity of the gene to be evolved in addition to the positive selection for a desired activity of the gene to be evolved. Such negative selection methods are useful, for example, in order to maintain enzyme specificity when increasing the efficiency of an enzyme directed towards a specific substrate. This can avoid, for example, the evolution of gene products that show a generally increased activity of the gene to be evolved, including an increased off-target activity, which is often undesired.

In some embodiments, negative selection is applied during a continuous evolution process as described herein, by penalizing the undesired activities of evolved gene products. This is useful, for example, if the desired evolved gene product is an enzyme with high specificity for a substrate or target site, for example, a transcription factor with altered, but not broadened, specificity. In some embodiments, negative selection of an undesired activity, e.g., off-target activity of the gene to be evolved, is achieved by causing the undesired activity to interfere with pIII production, thus inhibiting the propagation of phage genomes encoding gene products with an undesired activity. In some embodiments, expression of a dominant-negative version of pIII or expression of an antisense RNA complementary to the gIII RBS and/or gIII start codon is linked to the presence of an undesired activity of the gene to be evolved. Suitable negative selection strategies and reagents useful for negative selection, such as dominant-negative versions of M13 pIII, are described herein and in International PCT Application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. application, U.S. Ser. No. 13/922,812, filed Jun. 20, 2013; and U.S. application, U.S. Ser. No. 62/067,194, filed Oct. 22, 2014, the entire contents of each of which are incorporated herein by reference.

In some embodiments, counter-selection against undesired activity is achieved by linking such undesired activities to the inhibition of phage propagation. In some embodiments, a dual selection strategy is applied during a continuous evolution experiment, in which both positive selection and negative selection constructs are present in the host cells. In some such embodiments, the positive and negative selection constructs are situated on the same plasmid, also referred to as a dual selection accessory plasmid.

One advantage of using a simultaneous dual selection strategy is that the selection stringency can be fine-tuned based on the activity or expression level of the negative selection construct as compared to the positive selection construct. Another advantage of a dual selection strategy is that the selection is not dependent on the presence or the absence of a desired or an undesired activity, but on the ratio of desired and undesired activities, and, thus, the resulting ratio of pIII and pIII-neg that is incorporated into the respective phage particle.

For example, in some embodiments, the host cells comprise an expression construct encoding a dominant-negative form of the at least one gene for the generation of infectious phage particles, e.g., a dominant-negative form of the pI protein (pII-neg), under the control of an inducible promoter that is activated by a transcriptional activator other than the transcriptional activator driving the positive selection system. Expression of the dominant-negative form of the gene diminishes or completely negates any selective advantage an evolved phage may exhibit and thus dilutes or eradicates any variants exhibiting undesired activity from the lagoon.

Some aspects of this invention provide or utilize a dominant negative variant of pIII (pIII-neg). These aspects are based on the recognition that a pIII variant that comprises the two N-terminal domains of pIII and a truncated, termination-incompetent C-terminal domain is not only inactive but is a dominant-negative variant of pill. A pIII variant comprising the two N-terminal domains of pIII and a truncated, termination-incompetent C-terminal domain was described in Bennett, N. J.; Rakonjac, J., Unlocking of the filamentous bacteriophage virion during infection is mediated by the C domain of pIII. Journal of Molecular Biology 2006, 356 (2), 266-73; the entire contents of which are incorporated herein by reference. The dominant negative property of such pIII variants has been described in more detail in PCT Application PCT/US2011/066747, published as WO2012/088381 on Jun. 28, 2012, the entire contents of which are incorporated herein by reference.

The pIII-neg variant as provided in some embodiments herein is efficiently incorporated into phage particles, but it does not catalyze the unlocking of the particle for entry during infection, rendering the respective phage noninfectious even if wild type pIII is present in the same phage particle. Accordingly, such pIII-neg variants are useful for devising a negative selection strategy in the context of PACE, for example, by providing an expression construct comprising a nucleic acid sequence encoding a pIII-neg variant under the control of a promoter comprising a recognition motif, the recognition of which is undesired. In other embodiments, pIII-neg is used in a positive selection strategy, for example, by providing an expression construct in which a pII-neg encoding sequence is controlled by a promoter comprising a nuclease target site or a repressor recognition site, the recognition of either one is desired.

In some embodiments, the vector or phage encoding the gene to be evolved is a filamentous phage, for example, an M13 phage, such as an M13 selection phage as described in more detail elsewhere herein. In some embodiments, the host cells are cells amenable to infection by the filamentous phage, e.g., by M13 phage, such as, for example, E. coli cells. In some such embodiments, the gene required for the production of infectious viral particles is the M13 gene III (gIII) encoding the M13 protein III (pIII).

Typically, the vector/host cell combination is chosen in which the life cycle of the vector is significantly shorter than the average time between cell divisions of the host cell. Average cell division times and vector life cycle times are well known in the art for many cell types and vectors, allowing those of skill in the art to ascertain such host cell/vector combinations. In certain embodiments, host cells are being removed from the population of host cells in which the vector replicates at a rate that results in the average time of a host cell remaining in the host cell population before being removed to be shorter than the average time between cell divisions of the host cells, but to be longer than the average life cycle of the viral vector employed. The result of this is that the host cells, on average, do not have sufficient time to proliferate during their time in the host cell population while the viral vectors do have sufficient time to infect a host cell, replicate in the host cell, and generate new viral particles during the time a host cell remains in the cell population. This assures that the only replicating nucleic acid in the host cell population is the vector encoding the gene to be evolved, and that the host cell genome, the accessory plasmid, or any other nucleic acid constructs cannot acquire mutations allowing for escape from the selective pressure imposed.

For example, in some embodiments, the average time a host cell remains in the host cell population is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 120, about 150, or about 180 minutes.

In some embodiments, the average time a host cell remains in the host cell population depends on how fast the host cells divide and how long infection (or conjugation) requires. In general, the flow rate should be faster than the average time required for cell division, but slow enough to allow viral (or conjugative) propagation. The former will vary, for example, with the media type, and can be delayed by adding cell division inhibitor antibiotics (FtsZ inhibitors in E. coli, etc.). Since the limiting step in continuous evolution is production of the protein required for gene transfer from cell to cell, the flow rate at which the vector washes out will depend on the current activity of the gene(s) of interest. In some embodiments, titrable production of the protein required for the generation of infectious particles, as described herein, can mitigate this problem. In some embodiments, an indicator of phage infection allows computer-controlled optimization of the flow rate for the current activity level in real-time.

In some embodiments, a PACE experiment according to methods provided herein is run for a time sufficient for at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least, 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 7500, at least 10000, or more consecutive viral life cycles. In certain embodiments, the viral vector is an M13 phage, and the length of a single viral life cycle is about 10-20 minutes.

In some embodiments, the host cells are contacted with the vector and/or incubated in suspension culture. For example, in some embodiments, bacterial cells are incubated in suspension culture in liquid culture media. Suitable culture media for bacterial suspension culture will be apparent to those of skill in the art, and the invention is not limited in this regard. See, for example, Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press: 1989); Elizabeth Kutter and Alexander Sulakvelidze: Bacteriophages: Biology and Applications. CRC Press; 1st edition (December 2004), ISBN: 0849313368; Martha R. J. Clokie and Andrew M. Kropinski: Bacteriophages: Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions (Methods in Molecular Biology) Humana Press; 1st edition (December, 2008), ISBN: 1588296822; Martha R. J. Clokie and Andrew M. Kropinski: Bacteriophages: Methods and Protocols, Volume 2: Molecular and Applied Aspects (Methods in Molecular Biology) Humana Press; 1st edition (December 2008), ISBN: 1603275649; all of which are incorporated herein in their entirety by reference for disclosure of suitable culture media for bacterial host cell culture).

Suspension culture typically requires the culture media to be agitated, either continuously or intermittently. This is achieved, in some embodiments, by agitating or stirring the vessel comprising the host cell population. In some embodiments, the outflow of host cells and the inflow of fresh host cells is sufficient to maintain the host cells in suspension. This in particular, if the flow rate of cells into and/or out of the lagoon is high.

In some embodiments, the flow of cells through the lagoon is regulated to result in an essentially constant number of host cells within the lagoon. In some embodiments, the flow of cells through the lagoon is regulated to result in an essentially constant number of fresh host cells within the lagoon. Typically, the lagoon will hold host cells in liquid media, for example, cells in suspension in a culture media. However, lagoons in which adherent host cells are cultured on a solid support, such as on beads, membranes, or appropriate cell culture surfaces are also envisioned. The lagoon may comprise additional features, such as a stirrer or agitator for stirring or agitating the culture media, a cell densitometer for measuring cell density in the lagoon, one or more pumps for pumping fresh host cells into the culture vessel and/or for removing host cells from the culture vessel, a thermometer and/or thermocontroller for adjusting the culture temperature, as well as sensors for measuring pH, osmolarity, oxygenation, and other parameters of the culture media. The lagoon may also comprise an inflow connected to a holding vessel comprising a mutagen or a transcriptional inducer of a conditional gene expression system, such as the arabinose-inducible expression system of the mutagenesis plasmid described in more detail elsewhere herein.

In some embodiments, the host cell population is continuously replenished with fresh, uninfected host cells. In some embodiments, this is accomplished by a steady stream of fresh host cells into the population of host cells. In other embodiments, however, the inflow of fresh host cells into the lagoon is semi-continuous or intermittent (e.g., batch-fed). In some embodiments, the rate of fresh host cell inflow into the cell population is such that the rate of removal of cells from the host cell population is compensated. In some embodiments, the result of this cell flow compensation is that the number of cells in the cell population is substantially constant over the time of the continuous evolution procedure. In some embodiments, the portion of fresh, uninfected cells in the cell population is substantially constant over the time of the continuous evolution procedure. For example, in some embodiments, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, or about 90% of the cells in the host cell population are not infected by virus. In general, the faster the flow rate of host cells is, the smaller the portion of cells in the host cell population that are infected will be. However, faster flow rates allow for more transfer cycles, e.g., viral life cycles, and, thus, for more generations of evolved vectors in a given period of time, while slower flow rates result in a larger portion of infected host cells in the host cell population and therefore a larger library size at the cost of slower evolution. In some embodiments, the range of effective flow rates is invariably bounded by the cell division time on the slow end and vector washout on the high end In some embodiments, the viral load, for example, as measured in infectious viral particles per volume of cell culture media is substantially constant over the time of the continuous evolution procedure.

Typically, the fresh host cells introduced into the lagoon comprise a mutagenesis expression construct as provided herein, a selection system, e.g., an accessory plasmid encoding the at least one gene for the generation of infectious phage particles, and an expression construct providing other phage functions, such as, for example, a helper phage. In some embodiments, however, the host cells may not comprise an expression construct providing other phage functions, such as, for example, a helper phage, and those functions are provided to the host cells in some other way, e.g., as part of the selection phage. In some embodiments, the host cells are generated by contacting an uninfected host cell with the relevant vectors, for example, a vector comprising a mutagenesis expression construct as provided herein, an accessory plasmid, and, if desired, a helper phage, and growing an amount of host cells sufficient for the replenishment of the host cell population in a continuous evolution experiment. Methods for the introduction of plasmids and other gene constructs into host cells are well known to those of skill in the art and the invention is not limited in this respect. For bacterial host cells, such methods include, but are not limited to electroporation and heat-shock of competent cells. In some embodiments, the accessory plasmid comprises a selection marker, for example, an antibiotic resistance marker, and the fresh host cells are grown in the presence of the respective antibiotic to ensure the presence of the plasmid in the host cells. Where multiple plasmids are present, different markers are typically used. Such selection markers and their use in cell culture are known to those of skill in the art, and the invention is not limited in this respect.

In some embodiments, the host cell population in a continuous evolution experiment is replenished with fresh host cells growing in a parallel, continuous culture. In some embodiments, the cell density of the host cells in the host cell population contacted with the viral vector and the density of the fresh host cell population is substantially the same.

Typically, the cells being removed from the cell population contacted with the vector comprise cells that are infected with the vector and uninfected cells. In some embodiments, cells are being removed from the cell populations continuously, for example, by effecting a continuous outflow of the cells from the population. In other embodiments, cells are removed semi-continuously or intermittently from the population. In some embodiments, the replenishment of fresh cells will match the mode of removal of cells from the cell population, for example, if cells are continuously removed, fresh cells will be continuously introduced. However, in some embodiments, the modes of replenishment and removal may be mismatched, for example, a cell population may be continuously replenished with fresh cells, and cells may be removed semi-continuously or in batches.

In some embodiments, the rate of fresh host cell replenishment and/or the rate of host cell removal is adjusted based on quantifying the host cells in the cell population. For example, in some embodiments, the turbidity of culture media comprising the host cell population is monitored and, if the turbidity falls below a threshold level, the ratio of host cell inflow to host cell outflow is adjusted to effect an increase in the number of host cells in the population, as manifested by increased cell culture turbidity. In other embodiments, if the turbidity rises above a threshold level, the ratio of host cell inflow to host cell outflow is adjusted to effect a decrease in the number of host cells in the population, as manifested by decreased cell culture turbidity. Maintaining the density of host cells in the host cell population within a specific density range ensures that enough host cells are available as hosts for the evolving viral vector population, and avoids the depletion of nutrients at the cost of viral packaging and the accumulation of cell-originated toxins from overcrowding the culture.

In some embodiments, the cell density in the host cell population and/or the fresh host cell density in the inflow is about 102 cells/ml to about 1012 cells/ml. In some embodiments, the host cell density is about 102 cells/ml, about 103 cells/ml, about 104 cells/ml, about 105 cells/ml, about 5·10 cells/ml, about 106 cells/ml, about 5·106 cells/ml, about 107 cells/ml, about 5·107 cells/ml, about 108 cells/ml, about 5·108 cells/ml, about 109 cells/ml, about 5·109 cells/ml, about 1010 cells/ml, or about 5·1010 cells/ml. In some embodiments, the host cell density is more than about 1010 cells/ml.

The PACE methods provided herein are typically carried out in a lagoon. Suitable lagoons and other laboratory equipment for carrying out PACE methods as provided herein have been described in detail elsewhere. See, for example, International PCT Application, PCT/US2011/066747, published as WO2012/088381 on Jun. 28, 2012, the entire contents of which are incorporated herein by reference. In some embodiments, the lagoon comprises a cell culture vessel comprising an actively replicating population of vectors, for example, phage vectors comprising a gene of interest, and a population of host cells, for example, bacterial host cells. In some embodiments, the lagoon comprises an inflow for the introduction of fresh host cells into the lagoon and an outflow for the removal of host cells from the lagoon. In some embodiments, the inflow is connected to a turbidostat comprising a culture of fresh host cells. In some embodiments, the outflow is connected to a waste vessel, or a sink. In some embodiments, the lagoon further comprises an inflow for the introduction of a mutagen into the lagoon. In some embodiments that inflow is connected to a vessel holding a solution of the mutagen. In some embodiments, the lagoon comprises an inflow for the introduction of an inducer of gene expression into the lagoon, for example, of an inducer activating an inducible promoter within the host cells that drives expression of a gene promoting mutagenesis (e.g., as part of a mutagenesis plasmid), as described in more detail elsewhere herein. In some embodiments, that inflow is connected to a vessel comprising a solution of the inducer, for example, a solution of arabinose.

In some embodiments, the lagoon comprises a controller for regulation of the inflow and outflow rates of the host cells, the inflow of the mutagen, and/or the inflow of the inducer. In some embodiments, a visual indicator of phage presence, for example, a fluorescent marker, is tracked and used to govern the flow rate, keeping the total infected population constant. In some embodiments, the visual marker is a fluorescent protein encoded by the phage genome, or an enzyme encoded by the phage genome that, once expressed in the host cells, results in a visually detectable change in the host cells. In some embodiments, the visual tracking of infected cells is used to adjust a flow rate to keep the system flowing as fast as possible without risk of vector washout.

In some embodiments, the controller regulates the rate of inflow of fresh host cells into the lagoon to be substantially the same (volume/volume) as the rate of outflow from the lagoon. In some embodiments, the rate of inflow of fresh host cells into and/or the rate of outflow of host cells from the lagoon is regulated to be substantially constant over the time of a continuous evolution experiment. In some embodiments, the rate of inflow and/or the rate of outflow is from about 0.1 lagoon volumes per hour to about 25 lagoon volumes per hour. In some embodiments, the rate of inflow and/or the rate of outflow is approximately 0.1 lagoon volumes per hour (lv/h), approximately 0.2 lv/h, approximately 0.25 lv/h, approximately 0.3 lv/h, approximately 0.4 lv/h, approximately 0.5 lv/h, approximately 0.6 lv/h, approximately 0.7 lv/h, approximately 0.75 lv/h, approximately 0.8 lv/h, approximately 0.9 lv/h, approximately 1 lv/h, approximately 2 lv/h, approximately 2.5 lv/h, approximately 3 lv/h, approximately 4 lv/h, approximately 5 lv/h, approximately 7.5 lv/h, approximately 10 lv/h, or more than 10 lv/h.

In some embodiments, the inflow and outflow rates are controlled based on a quantitative assessment of the population of host cells in the lagoon, for example, by measuring the cell number, cell density, wet biomass weight per volume, turbidity, or cell growth rate. In some embodiments, the lagoon inflow and/or outflow rate is controlled to maintain a host cell density of from about 102 cells/ml to about 1012 cells/ml in the lagoon. In some embodiments, the inflow and/or outflow rate is controlled to maintain a host cell density of about 102 cells/ml, about 103 cells/ml, about 104 cells/ml, about 105 cells/ml, about 5×105 cells/ml, about 106 cells/ml, about 5×106 cells/ml, about 107 cells/ml, about 5×107 cells/ml, about 108 cells/ml, about 5×108 cells/ml, about 109 cells/ml, about 5×109 cells/ml, about 1010 cells/ml, about 5×1010 cells/ml, or more than 5×1010 cells/ml, in the lagoon. In some embodiments, the density of fresh host cells in the turbidostat and the density of host cells in the lagoon are substantially identical.

In some embodiments, the lagoon inflow and outflow rates are controlled to maintain a substantially constant number of host cells in the lagoon. In some embodiments, the inflow and outflow rates are controlled to maintain a substantially constant frequency of fresh host cells in the lagoon. In some embodiments, the population of host cells is continuously replenished with fresh host cells that are not infected by the phage. In some embodiments, the replenishment is semi-continuous or by batch-feeding fresh cells into the cell population.

In some embodiments, the lagoon volume is from approximately 1 ml to approximately 100 l, for example, the lagoon volume is approximately 1 ml, approximately 10 ml, approximately 50 ml, approximately 100 ml, approximately 200 ml, approximately 250 ml, approximately 500 ml, approximately 750 ml, approximately 11, approximately 2 l, approximately 2.5 l, approximately 3 l, approximately 4 l, approximately 5 l, approximately 10 l, approximately 20 l, approximately 50 l, approximately 75 l, approximately 100 l, approximately 1 ml-10 ml, approximately 10 ml-50 ml, approximately 50 ml-100 ml, approximately 100 ml-250 ml, approximately 250 ml-500 ml, approximately 500 ml-1 l, approximately 1 l-2 l, approximately 2 l-5 l, approximately 5 l-10 l, approximately 10 l-50 l, approximately 50 l-100 l, or more than 100 l.

In some embodiments, the lagoon and/or the turbidostat further comprises a heater and a thermostat controlling the temperature. In some embodiments, the temperature in the lagoon and/or the turbidostat is controlled to be from about 4° C. to about 55° C., preferably from about 25° C. to about 39° C., for example, about 37° C.

In some embodiments, the inflow rate and/or the outflow rate is controlled to allow for the incubation and replenishment of the population of host cells for a time sufficient for at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least, 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 7500, at least 10000, or more consecutive vector or phage life cycles. In some embodiments, the time sufficient for one phage life cycle is about 10, 15, 20, 25, or 30 minutes.

Therefore, in some embodiments, the time of the entire evolution procedure is about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 50 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about two weeks, about 3 weeks, about 4 weeks, or about 5 weeks.

In some embodiments, a PACE method as provided herein is performed in a suitable apparatus as described herein. For example, in some embodiments, the apparatus comprises a lagoon that is connected to a turbidostat comprising a host cell as described herein. In some embodiments, the host cell is an E. coli host cell. In some embodiments, the host cell comprises a mutagenesis expression construct as provided herein, an accessory plasmid as described herein, and, optionally, a helper plasmid as described herein, or any combination thereof. In some embodiments, the lagoon further comprises a selection phage as described herein, for example, a selection phage encoding a gene of interest. In some embodiments, the lagoon is connected to a vessel comprising an inducer for a mutagenesis plasmid, for example, arabinose. In some embodiments, the host cells are E. coli cells comprising the F′ plasmid, for example, cells of the genotype F′proA+B+Δ(lacIZY) zzf::Tn10(TetR)/endA 1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116λ−.

For example, in some embodiments, a PACE method as provided herein is carried out in an apparatus comprising a lagoon of about 100 ml, or about 1 l volume, wherein the lagoon is connected to a turbidostat of about 0.5 l, 1 l, or 3 l volume, and to a vessel comprising an inducer for a mutagenesis plasmid, for example, arabinose, wherein the lagoon and the turbidostat comprise a suspension culture of E. coli cells at a concentration of about 5×108 cells/ml. In some embodiments, the flow of cells through the lagoon is regulated to about 3 lagoon volumes per hour. In some embodiments, cells are removed from the lagoon by continuous pumping, for example, by using a waste needle set at a height of the lagoon vessel that corresponds to a desired volume of fluid (e.g., about 100 ml, in the lagoon. In some embodiments, the host cells are E. coli cells comprising the F′ plasmid, for example, cells of the genotype F′proA+B+Δ(lacIZY) zzf::Tn10(TetR)/endA1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116λ−.

In some embodiments, a mutagenesis plasmid (MP) comprises araC, dnaQ926, umuD′, umuC, and recA730, and is referred to as “MP1”. In some embodiments, a mutagenesis plasmid (MP) comprises araC and dnaQ926, and is referred to as “MP2”. In some embodiments, a mutagenesis plasmid (MP) comprises araC, dnaQ926, and dam, and is referred to as “MP3”. In some embodiments, a mutagenesis plasmid (MP) comprises araC, dnaQ926, dam, and seqA, and is referred to as “MP4”. In some embodiments, a mutagenesis plasmid (MP) comprises araC, dnaQ926, dam, seqA, ugi and pmCDA1, and is referred to as “MP5”. In some embodiments, a mutagenesis plasmid (MP) comprises araC, dnaQ926, dam, seqA, emrR, ugi and pmCDA1, and is referred to as “MP6”. In some embodiments, a mutagenesis plasmid comprises a sequence represented by any one of SEQ ID NO: 43-123. In some embodiments, a mutagenesis plasmid consists of a sequence represented by any one of SEQ ID NO: 43-123.

Modulation of Selection Stringency

Provided herein is a method for modulating the selection stringency in viral-assisted continuous evolution experiments. In some embodiments, the selection stringency is modulated by regulating the expression of a gene required for the generation of infectious viral particles (e.g., infectious phages). Generally, the gene required for the generation of infectious viral particles is on an accessory plasmid (AP) or on a drift plasmid (DP).

A drift plasmid allows evolutionary drift to take place to evolve weakly active or inactive gene variants. The expression of a gene required for the generation of infectious viral particles produces a protein required for the generation of infectious viral particles. In some embodiments, the gene required for the generation of infectious viral particles is gene III, which expresses the protein pIII needed to generate infections phage. In some embodiments, the modulation of the selection stringency is independent of the desired activity to be evolved. In some embodiments, regulation of the expression of the gene required for the generation of infectious viral particles is under the control of a small molecule inducible promoter (i.e., chemically-regulated promoters) and therefore, is dependent on the concentration of a small molecule. Examples of small molecule inducible promoters are known in the scientific literature (see, e.g., Yamamoto et. al., 2001, Neurobiology of Disease, 8: 923-932). Non-limiting examples of small molecule inducible promoters include lux promoters (e.g. Plux from vibrio fishceri induced by N-(3-oxohexanoyl)-L-homoserine lactone (OHHL)); alcohol-regulated promoters (e.g., alcohol dehydrogenase I promoter (alcA), lac promoter (e.g., Plac), arabinose-inducible promoters (e.g., Para), tetracycline-inducible promoters (e.g., Ptet), steroid-inducible promoters, and tamoxifen-inducible promoters. In some embodiments, the small molecule inducible promoter is a TetA promoter (Ptet). In some embodiments, the small molecule is tetracycline or tetracycline analogs. In some embodiments, the small molecule is anhydrotetracycline (ATc). In some embodiments, the small molecule is doxycycline. In some embodiments, the host cell drift promoter is partly a tetracycline-inducible promoter (Ptet), which drives expression of the TetR repressor and TetA, the protein that pumps tetracycline out of the cell. In the absence of tetracycline or its analogs, TetR binds to the TetR operator sites and prevents transcription. In the presence of tetracycline or its analogs. TetR binds to tetracycline or a tetracycline analog, which induces a conformational change, making it unable to interact with the operator, so that target gene expression can occur.

In some embodiments, the host cell becomes viral infection-resistant prior to encountering the viral particle, thereby preventing viral propagation. For example, low levels of pill, such as the levels expressed at the beginning of a PACE experiment, have been shown to render cells resistant to filamentous phage infection.10 Accordingly, for situations where low levels of the protein (e.g., pIII) required for the generation of infectious viral particles renders host cells resistant to viral infection, it may be desirable to make the expression of the protein (e.g., pIII) required for the generation of infectious viral particles to be dependent on the condition that there be a prior viral infection of the host cells. In some embodiments, an E. coli phage shock promoter (Ppsp) is used to require prior viral infection. Transcription from Ppsp is induced by infection with filamentous phage via a pIV-dependent signaling cascade11 or by overexpression of a plasmid-encoded phage pIV gene.

To produce a system in which protein expression requires both the presence of the small molecule and prior viral infection, provided herein is a drift promoter. In some embodiments, the drift promoter is located on a drift plasmid in the host cell. In some embodiments, the drift promoter is produced from a Ppsp variant with a TetR operator installed at a position to disrupt either PspF or E. coli RNA polymerase binding. In some embodiments, the TetR operator is placed adjacent to the +1 transcription initiation site to produce a host cell drift promoter called Ppsp-tet which is induced only with the combination of phage infection and ATc. In some embodiments, the Ppsp-tet is placed upstream of the gene encoding the pIII protein. In some embodiments, propagation of the viral vector (e.g., SP) proceeds without activity-dependent gene III expression. In some embodiments, SPs can propagate in a small-molecule-dependent, activity-independent manner using the host cell drft promoter. In some embodiments, the drift promoter is produced from one that is activated upon pspF release after phage infection. In some embodiments, the host cell drift promoter is produced from another promoter such as ones upstream of pal or hyfR.

In some embodiments, a drift plasmid (DP) comprises an expression construct in which a drift promoter drives expression of a gene required for the generation of infectious viral particles (e.g., gIII), and an expression construct comprising a sequence encoding one or more gene product(s) that increase(s) the mutation rate in a host cell, e.g., in a bacterial host cell. The one or more gene product(s) that increase(s) the mutation rate in a host cell is, in some embodiments, araC, dnaQ926, umuD′, umuC, recA730, dam, seqA, emrR, PBS2, UGI, or pmCDA1, or any combination thereof. In some embodiments, a drift plasmid (DP) comprises an expression construct in which a drift promoter drives expression of a gene required for the generation of infectious viral particles (e.g., gIII), wherein the expression construct is on the same plasmid as a mutagenesis expression construct provided herein, e.g., a mutagenesis expression construct provided in the context of the mutagenesis plasmids provided herein.

In some embodiments, a drift plasmid (DP) comprises araC, dnaQ926, umuD′, umuC, recA730, and an anhydrotetracycline (ATc)-dependent drift promoter, and is referred to as “DP1”. In some embodiments, the ATc-dependent promoter drives expression of a gene required for the generation of infectious viral particles (e.g., gIII). One embodiment of a DP1 plasmid is shown in FIG. 20, and is represented by SEQ ID NO: 27.

In some embodiments, a drift plasmid (DP) comprises araC, dnaQ926, and an anhydrotetracycline (ATc)-dependent drift promoter, and is referred to as “DP2”. In some embodiments, the ATc-dependent promoter drives expression of a gene required for the generation of infectious viral particles (e.g., gIII). One embodiment of a DP2 plasmid is shown in FIG. 21, and is represented by SEQ ID NO: 28.

In some embodiments, a drift plasmid (DP) comprises araC, dnaQ926, dam, and an anhydrotetracycline (ATc)-dependent drift promoter, and is referred to as “DP3”. In some embodiments, the ATc-dependent promoter drives expression of a gene required for the generation of infectious viral particles (e.g., gIII). One embodiment of a DP3 plasmid is shown in FIG. 22, and is represented by SEQ ID NO: 29.

In some embodiments, a drift plasmid (DP) comprises araC, dnaQ926, dam, seqA, and an anhydrotetracycline (ATc)-dependent drift promoter, and is referred to as “DP4”. In some embodiments, the ATc-dependent promoter drives expression of a gene required for the generation of infectious viral particles (e.g., gIII). One embodiment of a DP4 plasmid is shown in FIG. 23, and is represented by SEQ ID NO: 33.

In some embodiments, a drift plasmid (DP) comprises araC, dnaQ926, dam, seqA, ugi and pmCDA1, and an anhydrotetracycline (ATc)-dependent drift promoter, and is referred to as “DP5”. In some embodiments, the ATc-dependent promoter drives expression of a gene required for the generation of infectious viral particles (e.g., gIII). One embodiment of a DP5 plasmid is shown in FIG. 24, and is represented by SEQ ID NO: 34.

In some embodiments, a drift plasmid (DP) comprises araC, dnaQ926, dam, seqA, emrR, ugi and pmCDA1, and an anhydrotetracycline (ATc)-dependent drift promoter, and is referred to as “DP6”. In some embodiments, the ATc-dependent promoter drives expression of a gene required for the generation of infectious viral particles (e.g., gIII). One embodiment of a DP6 plasmid is shown in FIG. 25, and is represented by SEQ ID NO: 35.

In some embodiments, provided is a method of tuning the selection stringency in continuous directed evolution methods. For example, to tune the selection stringency, a host cell can use the following plasmids: an activity-dependent AP, such as a PT7-gene III AP, in which gene II is controlled by an activity-dependent promoter, and a drift plasmid (DP) with a host cell drift promoter-gene III cassette, such as a Ppsp-tet gene III. In some embodiments, the AP additionally contains a reporter gene such as a luciferase gene. In some embodiments, the selection stringency is inversely proportional to the concentration of the small molecule used. In some embodiments, low selection stringency conditions are used. For example, saturating amounts of a small molecule inducer (e.g., ATc) allows the Ppsp-tet-gene III cassette in the DP to provide sufficient pII to maximize phage propagation, regardless of the SP-encoded property (such as activity), thus enabling genetic drift of the SP (low stringency). In some embodiments, an intermediate selection stringency is used. For example, at intermediate concentrations of a small molecule inducer (e.g., ATc), SPs encoding active library members have a replicative advantage over an SP encoding a weakly active/inactive variant by inducing additional pIII expression from an activity-dependent manner. An intermediate concentration is determined by sampling a number of concentrations of the small molecule by using a plasmid the carries the native Ptet promoter driving bacterial luciferase. Intermediate concentrations are typically considered those around the inflection point of a sigmoidal graph. In some embodiments, high selection stringency conditions are used. For example, a zero or low amount of a small molecule inducer (e.g., ATc) allows the selection stringency to be determined by the activity-dependent AP with no assistance from the Ppsp-tet-gene III cassette (high stringency).

In some embodiments, the evolution experiments uses a ratio of SPs with active starting genetic libraries to SPs with weakly active/inactive starting genetic libraries of about 1:1, 1:5, 1:10, 1:20, 1:40, 1:60, 1:80, 1:100, 1:120, 1:60, or 1:200. In some embodiments, the ratio of SPs with active to weakly active/inactive starting libraries is 1:100. In some embodiments, phage population is generally followed over time using a detectable label or directly via standard techniques. For example, the phage population can be followed using a combination of restriction endonuclease digests and/or real-time measurements of luminescence monitoring of promoter transcriptional activity (e.g., PT7 transcriptional activity), as further described herein. Additional methods are PCR, plaque assays, analysis by gel electrophoresis, or analytical digestion. In some embodiments, an accessory plasmid carrying the gene III and a gene encoding a co-expressed reporter fluorescent protein (such as the luciferase gene, GFP, or other fluorescent protein described herein) under the control of a conditional promoter (such as a PT7 or PT3) would produce luminescence from the translated luciferase when there is promoter transcriptional activity.

Selection stringency modulation can be used at any point in the continuous evolution process. In some embodiments, selection stringency modulation is used towards the end of the continuous evolution process. In some embodiments, selection stringency modulation is used towards the beginning of the continuous evolution process. In some embodiments, the selection stringency modulation is combined with negative selection.

In an embodiment, provided is a method for modulating the selection stringency during viral-assisted evolution of a gene product, the method comprising: (a) introducing host cells into a lagoon, wherein the host cell comprises a low selection stringency plasmid and a high selection stringency plasmid, wherein the low selection stringency plasmid comprises a viral gene required to package the selection viral vector into an infectious viral particles, wherein at least one gene required to package the selection viral vector into an infectious viral particles is expressed in response to the a concentration of a small molecule, and wherein the high selection stringency plasmid comprises a second copy of the viral gene required to package the selection viral vector into the infectious viral particles, wherein at least one viral gene required to package the selection viral vector into an infectious viral particles is expressed in response to a desired activity property of a gene product encoded by the gene to be evolved or an evolution product thereof; (b) introducing a selection viral vector comprising a gene to be evolved into a flow of host cells through a lagoon, wherein the gene to be evolved produces an active gene product or a weakly active or inactive gene product, wherein the active gene product has an activity that drives the expression of the viral gene required to package the selection viral vector into infectious viral particles in the high selection stringency plasmid and wherein the weakly active or inactive gene product has a relatively lower activity than the activity of the active gene product; and (c) mutating the gene to be evolved within the flow of host cells, wherein the host cells are introduced through the lagoon at a flow rate that is faster than the replication rate of the host cells and slower than the replication rate of the virus thereby permitting replication of the selection viral vector in the lagoon. In an embodiment, the host cells are fed from a chemostat into the lagoon.

In some embodiments, the method further comprising isolating the selection viral vector comprising an evolved product from the flow of cells and determining one or more properties of the evolved product. In some embodiments, the low selection stringency plasmid contains a drift promoter that is activated by a concentration of a small molecule inducer and/or prior viral infection. In one embodiment, the high selection stringency plasmid contains a promoter that is activated by a desired property of a gene product encoded by the gene to be evolved or an evolution product thereof. In yet another embodiment, the low selection stringency plasmid comprises a mutagenesis cassette under the control of a small-molecule inducible promoter. In another embodiment, the low selection stringency plasmid allows a high level of evolutionary drift to occur when the drift promoter is activated in response to a concentration of a small molecule inducer and/or prior viral infection. In some embodiments, the high selection stringency plasmid allows a low level of evolutionary drift to occur when the promoter is activated in response to a desired activity property of a gene product encoded by the gene to be evolved or an evolution product thereof.

In some embodiments, the property of the gene to be evolved originated from a weakly active or inactive starting gene. In some embodiments, the property of the gene to be evolved originated from an active starting gene. In an embodiment, the high selection stringency comprises a T7 promoter. In another embodiment, the low selection stringency comprises a drift promoter that is activated by a small-molecule inducer and/or prior viral infection. In some embodiments, the drift promoter is a Ppsp-tet promoter.

In some embodiments, the method of modulating the selection stringency further comprises the use of negative selection and/or positive selection.

Host Cells

Some aspects of this invention relate to host cells for continuous evolution processes as described herein. In some embodiments, a host cell is provided that comprises a mutagenesis expression construct as provided herein. In some embodiments, the host cell further comprises additional plasmids or constructs for carrying out a PACE process, e.g., a selection system comprising at least one viral gene encoding a protein required for the generation of infectious viral particles under the control of a conditional promoter the activity of which depends on a desired function of a gene to be evolved. For example, some embodiments provide host cells for phage-assisted continuous evolution processes, wherein the host cell comprises an accessory plasmid comprising a gene required for the generation of infectious phage particles, for example, M13 gIII, under the control of a conditional promoter, as described herein. In some embodiments, the host cell further provides any phage functions that are not contained in the selection phage, e.g., in the form of a helper phage. In some embodiments, the host cell provided further comprises an expression construct comprising a gene encoding a mutagenesis-inducing protein, for example, a mutagenesis plasmid as provided herein.

In some embodiments, modified viral vectors are used in continuous evolution processes as provided herein. In some embodiments, such modified viral vectors lack a gene required for the generation of infectious viral particles. In some such embodiments, a suitable host cell is a cell comprising the gene required for the generation of infectious viral particles, for example, under the control of a constitutive or a conditional promoter (e.g., in the form of an accessory plasmid, as described herein). In some embodiments, the viral vector used lacks a plurality of viral genes. In some such embodiments, a suitable host cell is a cell that comprises a helper construct providing the viral genes required for the generation of infectious viral particles. A cell is not required to actually support the life cycle of a viral vector used in the methods provided herein. For example, a cell comprising a gene required for the generation of infectious viral particles under the control of a conditional promoter may not support the life cycle of a viral vector that does not comprise a gene of interest able to activate the promoter, but it is still a suitable host cell for such a viral vector.

In some embodiments, the host cell is a prokaryotic cell, for example, a bacterial cell. In some embodiments, the host cell is an E. coli cell. In some embodiments, the host cell is a eukaryotic cell, for example, a yeast cell, an insect cell, or a mammalian cell. The type of host cell, will, of course, depend on the viral vector employed, and suitable host cell/viral vector combinations will be readily apparent to those of skill in the art.

In some embodiments, the viral vector is a phage and the host cell is a bacterial cell. In some embodiments, the host cell is an E. coli cell. Suitable E. coli host strains will be apparent to those of skill in the art, and include, but are not limited to, New England Biolabs (NEB) Turbo, Top10F′, DH12S, ER2738, ER2267, and XL1-Blue MRF′. These strain names are art recognized and the genotype of these strains has been well characterized. It should be understood that the above strains are exemplary only and that the invention is not limited in this respect.

In some PACE embodiments, for example, in embodiments employing an M13 selection phage, the host cells are E. coli cells expressing the Fertility factor, also commonly referred to as the F factor, sex factor, or F-plasmid. The F-factor is a bacterial DNA sequence that allows a bacterium to produce a sex pilus necessary for conjugation and is essential for the infection of E. coli cells with certain phage, for example, with M13 phage. For example, in some embodiments, the host cells for M13-PACE are of the genotype F′proA+B+Δ(lacIZY) zzf::Tn10(TetR)/endA1 recA1 galE15 galK16 nupG rpsL ΔlaciZYA araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116λ.

Kits and Apparatuses

Some aspects of this disclosure provide kits comprising (a) an expression construct or plasmid as provided herein, wherein the expression construct comprises an inducible promoter controlling at least one of the nucleic acid sequences comprised in the expression construct; and (b) an inducing agent that induces expression from the inducible promoter. In some embodiments, the kit further comprises (c) a vector encoding an M13 phage backbone and a multiple cloning site for insertion of a nucleic acid sequence encoding a gene product to be evolved, wherein the vector or a replication product thereof can be packaged into infectious phage particles in the presence of other phage functions by suitable host cells, but lacks at least one gene required for the generation of infectious particles. In some embodiments, the kit further comprises (d) an accessory plasmid comprising a nucleic acid sequence encoding the at least one gene required for the generation of infectious particles under the control of a promoter that is activated by a desired activity of the gene product to be evolved. In some embodiments, the kit further comprises (e) an accessory plasmid comprising a nucleic acid sequence encoding a dominant-negative version of the at least one gene required for the generation of infectious particles under the control of a promoter that is activated by an undesired activity of the gene product to be evolved. In some embodiments, the kit further comprises a helper phage providing all phage functions except for the at least one gene required for the generation of infectious phage particles provided by the accessory plasmid of (d). In some embodiments, the helper phage or a replication product thereof cannot be packaged into infectious phage particles. In some embodiments, the kit comprises suitable host cells. In some embodiments, the host cells are E. coli host cells.

Some aspects of this invention provide kits for continuous directed evolution as described herein. In some embodiments, the kit comprises (a) a vector encoding a phage backbone, for example, an M13 phage backbone, and a multiple cloning site for insertion of a nucleic acid sequence encoding a gene of interest. In some embodiments, the vector or a replication product thereof can be packaged into infectious phage particles in the presence of other phage functions by suitable host cells. In some embodiments, the vector or a replication product thereof lacks at least one gene required for the generation of infectious particles.

In some embodiments, the kit comprises (b) an accessory plasmid comprising a nucleic acid sequence encoding the at least one gene required for the generation of infectious particles under the control of a conditional promoter that is activated by a transcriptional activator.

In some embodiments, the kit further comprises a helper phage providing all phage functions except for the at least one gene required for the generation of infectious phage particles provided by the accessory plasmid of (b). In some embodiments, the helper phage or a replication product thereof cannot be packaged into infectious phage particles.

In some embodiments, the kit comprises suitable host cells. In some embodiments, the host cells are E. coli host cells. In some embodiments, the kit further comprises a mutagenesis plasmid. In some embodiments, the mutagenesis plasmid comprising a gene expression cassette encoding umuC (a components of E. coli translesion synthesis polymerase V), dam (deoxyadenosine methylase), and/or seqA (a hemimethylated-GATC binding domain), or any combination thereof.

In some embodiments, a PACE apparatus is provided, comprising a lagoon that is connected to a turbidostat comprising a host cell as described herein. In some embodiments, the host cell is an E. coli host cell. In some embodiments, the host cell comprises a mutagenesis expression construct as described herein, an accessory plasmid as described herein, and optionally, a helper plasmid as described herein, or any combination thereof. In some embodiments, the lagoon further comprises a selection phage as described herein, for example, a selection phage encoding a gene of interest. In some embodiments, the lagoon is connected to a vessel comprising an inducer for a mutagenesis plasmid, for example, arabinose. In some embodiments, the host cells are E. coli cells comprising the F′ plasmid, for example, cells of the genotype F′proA+B+ Δ(lacIZY) zzf::Tn10(TetR)/endA1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araDl39 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116λ−.

For example, in some embodiments, a PACE apparatus is provided, comprising a lagoon of about 100 ml, or about 1 l volume, wherein the lagoon is connected to a turbidostat of about 0.5 l, 1 l, or 3 l volume, and to a vessel comprising an inducer for a mutagenesis plasmid, for example, arabinose, wherein the lagoon and the turbidostat comprise a suspension culture of E. coli cells at a concentration of about 5×108 cells/ml. In some embodiments, the flow of cells through the lagoon is regulated to about 3 lagoon volumes per hour. In some embodiments, cells are removed from the lagoon by continuous pumping, for example, by using a waste needle set at a height of the lagoon vessel that corresponds to a desired volume of fluid (e.g., about 100 ml, in the lagoon. In some embodiments, the host cells are E. coli cells comprising the F′ plasmid, for example, cells of the genotype F′proA+B+Δ(lacIZY) zzf::Tn10(TetR)/endA1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116)λ−.

Some of the embodiments, advantages, features, and uses of the technology disclosed herein will be more fully understood from the Examples below. The Examples are intended to illustrate some of the benefits of the present disclosure and to describe particular embodiments, but are not intended to exemplify the full scope of the disclosure and, accordingly, do not limit the scope of the disclosure.

EXAMPLES

The potency of some exemplary mutagenesis systems provided herein was characterized, as well as the resulting mutational spectrum. Importantly, this system outperforms commonly used in vivo (XL1-Red, mutA, and LF-Pol I strains) and chemical (EMS, MNNG, and 2AP) methods in mutational efficiency and spectrum, and can even achieve mutational potency and spectrum comparable to that of state-of-the-art in vitro mutagenesis methods (such as PCR mutagenesis with Mutazyme II and Taq polymerase variants). We applied this system to two directed evolution case studies in bacteria and bacteriophage, validating their ability to access biomolecule variants with novel properties in shorter time frames and with greater effectiveness than previously described methods. Collectively, this system substantially advances in vivo mutagenesis capabilities and increases the effectiveness of laboratory evolution efforts.

Results Mutagenesis Plasmid Minimization

Bacteria control the rate of chromosomal substitutions through a series of overlapping mechanisms that can be subdivided into three main pathways: proofreading (reduces mutation rate by a factor of ˜102 substitutions/bp/generation), mismatch repair (reduces mutation rate by ˜103 substitutions/bp/generation), and base selection (reduces mutation rate by ˜105 substitutions/bp/generation) [10] (FIG. 7). These redundant replication maintenance mechanisms collectively account for the basal substitution rate of bacterial chromosomal DNA of ˜10−9 to 10−10 substitutions/bp/generation [10]. Based on prior knowledge of dominant mutators alleles that interfere with DNA replication fidelity, we sought to design a series of small-molecule inducible mutagenesis plasmids (MPs) that offer broad mutational spectra and high levels of mutagenesis in bacterial cells.

We recently reported the development and application of an MP for in vivo mutagenesis during phage-assisted continuous evolution (PACE) [11]. This MP increases the mutation rate of the M13 bacteriophage ˜100-fold above the basal E. coli mutation rate through the arabinose-induced expression of dnaQ926, a dominant negative variant of the E. coli DNA Pol 11H proofreading domain. This plasmid additionally provides umuD′, umuC and recA730, which together allow for in vivo translesion mutagenesis employing UV light or chemical mutagens. This MP (designated MP1) results in a substitution rate of 7.2×10−5 and 5.4×10−8 substitutions/bp/generation for M13 phage and E. coli, respectively [11, 12]. This mutation rate, however, is still several orders of magnitude below the mutation rates provided by conventional in vitro mutagenesis techniques [13].

Since we sought to avoid the use of exogenous mutagens, we first minimized MP1 by removing umuD′, umuC, and recA730 from MP1 to yield MP2 carrying only dnaQ926, and observed mutation rates in the absence of mutagens to be modestly improved compared to MP1 through a rifampin-resistance assay using the nearly wild-type E. coli MG1655 ΔrecA::apra (Table 1), enabling an average of 3.6×10−7 substitutions/bp/generation (FIG. 1A). Since dnaQ926 abrogates the proofreading component of DNA replication, we began assessing additional genes that when expressed from the MP can further enhance mutation rate.

TABLE 1 Summary of all strains used in this study. Strains that were requested from the Yale Coli Genetic Stock Center (CGSC) show the corresponding strain numbers. Strain CGSC # Genotype MG1655 12492 F− ΔrecA1918::apra, rph-1 λ− ΔrecA CSH101 8095 F′ lacI373 lacZ571/ara-600 Δ(gpt-lac)5 relA1 spoT1 thiE1 λ− CSH102 8096 F′ lacI373 lacZ572/ara-600 Δ(gpt-lac)5 relA1 spoT1 thiE1 λ− CSH103 8097 F′ lacI373 lacZ573/ara-600 Δ(gpt-lac)5 relA1 spoT1 thiE1 λ− CSH104 8098 F′ lacI373 lacZ574/ara-600 Δ(gpt-lac)5 relA1 spoT1 thiE1 λ− CSH105 8099 F′ lacI373 lacZ575/ara-600 Δ(gpt-lac)5 relA1 spoT1 thiE1 λ− CSH106 8100 F′ lacI373 lacZ576/ara-600 Δ(gpt-lac)5 relA1 spoT1 thiE1 λ− S1030 N/A F' proA + B + Δ(lacIZY) zzf.:Tn10 lacIQ1 PN25-tetR luxCDE/endA1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara, leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116 araE201 ΔrpoZ Δflu ΔcsgABCDEFG ΔpgaC λ− S1021 N/A F− endA1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara, leu)7697 mcrA Δ(mrr-hsdRMS- mcrBC) proBA::pir116 araE201 ΔrpoZ Δflu ΔcsgABCDEFG ΔpgaC λ−

DNA Methylation State Manipulation Enhances Mutagenesis

Mismatch repair reduces the error rate of bacterial DNA replication by a factor of up to 103 [10]. Following daughter strand synthesis, MutSL scan the genome for mismatches that have evaded the base selection and proofreading activities of the E. coli DNA Pol III holoenzyme (FIG. 7). Once recognized, the newly synthesized DNA is nicked by MutH at hemimethylated GATC sequences, then unwound and digested by dedicated helicases and exonucleases. DNA Pol Iii synthesizes a new strand and Dam methylase methylates the resulting DNA. The deletion of mutS, mutL, or mutH, and the overexpression of dam are known to have a strong mutator effect due to impaired mismatch repair [14].

We added wild-type dam downstream of dnaQ926 on the same arabinose-inducible expression cassette to yield MP3, and observed increased mutagenesis potency but also increased uninduced background mutagenesis, likely due to a strong cryptic σ70 promoter at the 3′ end of dnaQ926 (FIG. 8). Overall, MP3 resulted in a 3-fold mutagenesis potency increase in the presence of arabinose relative to MP2 but >60-fold increase in background mutagenesis, greatly reducing the dynamic range of induced mutagenesis to only 11-fold (FIG. 1A).

To restore dynamic range, we installed the gene encoding the hemimethylated GATC-binding domain SeqA downstream of dam. Low-level expression of seqA is known to delay Dam methylation of hemimethylated GATC sequences [15] and induce positive supercoils in chromosomal and episomal DNA [16], which may reduce MP gene transcription in the absence of arabinose. Conversely, high-level seqA expression results in a potent mutator response [17] and induction of negative supercoils in chromosomal and episomal DNA [16], potentially increasing global gene transcription, including that of the MP, upon induction with arabinose. Indeed, the MP carrying dnaQ926, dam, and seqA (MP4) resulted in >60-fold reduced background and 2-fold improved mutagenesis potency in the presence of arabinose relative to MP3, representing a cumulative >100-fold improvement in dynamic range (FIG. 1A).

While the addition of dam and seqA together increased the mutagenic potency by only 4-fold compared to MP2, it is known that dominant-negative dnaQ mutants partially saturate the mismatch repair response [18], Indeed, including dominant-negative variants of mutS 117, 191, mutL [20, 21], or mutH [21] with dnaQ926 had no additional effect on mutagenesis (Tables 2 and 3). Taken together, these results suggest that the inclusion of seqA and dam on MP4 is sufficient to fully disrupt the mismatch repair response, enabling an average of 4.4×10˜7 substitutions/bp/generation by the rifampicin resistance assay.

TABLE 2 Summary of all ORFs carried by the MPs. All MPs use the identical vector backbone: a cloDFI3 origin of replication (20-40 copies/cell), a chloramphenicol resistance cassette, the arabinose responsive promoter PBAD driving the imitator genes, and the weak promoter PC driving araC. Genes carried by each MP are arranged in the order found in the table and are highlighted according to their mechanism of action and/or the canonical repair pathway that they disrupt: proofreading (blue), translesion synthesis (purple), methyl- directed mismatch repair (red), base excision repair (green), base selection (yellow) and unknown (black). Additional optimizations included: codon usage optimization (opt) and increased ribosome-binding site strength (*). Boxes are not drawn to scale. Nucleotide and protein sequences related to the gene symbols listed in the table above are known to those of skill in the art. For gene symbols for which publications are indicated (by superscript numbers), any sequences provided in the publication related to the gene symbol is incorporated herein by reference. In some embodiments, the gene symbols above relate to nucleotide and protein sequences accessible under that gene symbol in any of the National Center for Biotechnology Information (NCBI) databases, for example, in the Nucleotide Reference Sequence (RefSeq) database, release 69 (Jan. 2, 2015), or in the Unigene database available at the time of filing, and any NCBI database entries, e.g., RefSeq database entries for the listed genes in the ResSeq database, release 69, or in the Unigene database at the time of filing, are incorporated herein by reference. Name Genes encoded on the MP (native order) MP1 dnaQ92640 umuD′ umuC recA73030 MP2 dnaQ926 MP-B2 dnaE7442 MP-B4 dnaE48624 MP-B5 dnaE102643 MP-C2 dnaX3645 MP-C3 dnaX201645 MP-D3 dnaQ926 dnaE486 MP-D4 dnaQ926 dnaE1026 MP-E dnaQ926 dnaX36 MP-E2 dnaQ926 dnaX2016 MP-F2 dnaQ926 mutS53819 MP-F3 dnaQ926 mutS50319 MP-H dnaQ926 mutL705220 MP-H2 dnaQ926 mutL71320 MP-H3 dnaQ926 mutL (R261H)21 MP-H4 dnaQ926 mutL (K307A)21 MP-I dnaQ926 mutH (E56A)21 MP-I2 dnaQ926 mutH (K79E)21 MP-I3 dnaQ926 mutH (K116E)21 MP-J rpsD1247 MP-J2 rpsD1447 MP-J3 rpsD1647 MP3 dnaQ926 dam MP-K7 dnaQ926 dam emrR17 MP4 dnaQ926 dam seqA17 MP-K9 dnaQ926 dam mutSΔN17 MP-K10 dnaQ926 dam seqA emrR MP-K11 dnaQ926 dam seqA mutSΔN MP-K12 dnaQ926 dam seqA dinB42 MP-K13 dnaQ926 dam seqA polB52 MP-K14 dnaQ926 dam seqA* MP-L polB MP-L2 polB (D156A)52 MP-P dnaQ926 dam seqA emrR mutH (ES6A) MP-P3 dnaQ926 dam seqA emrR mutL713 MP-P4 dnaQ926 dam seqA emrR mutS503 MP-P5 dnaQ926 dam seqA emrR mutSΔN MP-P6 dnaQ926 dam seqA emrR dinB MP-P7 dnaQ926 dam seqA emrR polB MP-P8 dnaQ926 dam seqA emrR Ugi41 AID22 MP-P9 dnaQ926 dam seqA emrR Ugi APOBEC122 MP6 dnaQ926 dam seqA emrR Ugi CDA122 MP-P11 dnaQ926 dam seqA emrR Ugi CDA1 mutSΔN MP-Q dnaQ926 dam seqA rsmE44 MP-Q2 dnaQ926 dam seqA cchA44 MP-Q3 dnaQ926 dam seqA yffl41 MP-Q4 dnaQ926 dam seqA yfjY17 MP-Q5 dnaQ926 dam seqA ugi AID MP-Q6 dnaQ926 dam seqA ugi APOBEC1 MP5 dnaQ926 dam seqA ugi CDA1 MP-Q8 dnaQ926 dam seqA nrdAB25 MP-Q9 dnaQ926 dam seqA nrdA (H59A)B25 MP-Q10 dnaQ926 dam seqA nrdA (A65V)B46 MP-Q11 dnaQ926 dam seqA nrdA (A301V)B46 MP-Q12 dnaQ926 dam seqA nrdAB (P334L)46 MP-Q13 dnaQ926 dam seqA nrdEF25 MP-R dnaQ926 dam seqA ugi AID (opt) MP-R2 dnaQ926 dam seqA ugi APOBEC1 (opt) MP-R3 dnaQ926 dam seqA ugi CDA1 (opt) MP-R4 dnaQ926 dam seqA emrR ugi AID (opt) MP-R5 dnaQ926 dam seqA emrR ugi APOBEC1 (opt) MP-R6 dnaQ926 dam seqA emrR ugi CDA1 (opt) MP-S dnaQ926 MAG149 MP-S2 dnaQ926 AAG (Y127I-H136L)50 MP-S3 dnaQ926 Δ80-AAG (Y127I-H136L)50 MP-T dnaQ926 dam seqA emrR ugi AID (7)51 MP-T2 dnaQ926 dam seqA emrR ugi AID (73)51 MP-T3 dnaQ926 dam seqA emrR ugi AID (7.33)51 MP-T4 dnaQ926 dam seqA emrR ugi AID (7.3.3)51 MP-T5 dnaQ926 dam seqA emrR ugi AID (7.3.1)51 MP-T6 dnaQ926 dam seqA emrR ugi AID (7.3.2)51 MP-U dnaQ926* dam seqA emrR ugi CDA1 MP-U2 dnaQ926 dam* seqA emrR ugi CDA1 MP-U3 dnaQ926 dam seqA emrR* ugi CDA1 MP-U4 dnaQ926 dam seqA emrR ugi CDA1* MP-V BR1353 dam seqA emrR ugi CDA1 MP-V2 BRM153 dam seqA emrR ugi CDA1 MP-V3 BR1153 dam seqA emrR ugi CDA1 MP-V4 BR653 dam seqA emrR ugi CDA1 MP-V5 BR153 dam seqA emrR ugi CDA1

TABLE 3 Summary of induced and uninduced mutagenesis levels for all designed MPs. All MPs were tested using the rifampin resistance assay to assess their relative mutagenic load under uninduced (glucose) and induced (arabinose) conditions. The viability of the MP-carrying strains under the induced conditions (as a percentage of the viability of the strain without an MP) is also shown. Ideal MPs show low background and high induced mutagenesis, with only moderate reductions in viability. Name Uninduced μbp Induced μbp Viability (%) None 1.20E−10 2.20E−11 100 MP1 3.00E−09 2.40E−07 87.5 MP2 1.00E−09 3.60E−07 136.8 MP-B2 0.00E+00 0.00E+00 25.4 MP-B4 0.00E+00 0.00E+00 45.8 MP-B5 0.00E+00 0.00E+00 55.9 MP-C2 0.00E+00 0.00E+00 63.6 MP-C3 0.00E+00 0.00E+00 53.4 MP-D3 0.00E+00 8.50E−09 24.2 MP-D4 0.00E+00 9.50E−09 68.6 MP-E 0.00E+00 7.80E−10 61 MP-E2 1.50E−09 2.50E−09 40.7 MP-F2 3.60E−09 9.80E−09 48.3 MP-F3 0.00E+00 1.70E−08 73.7 MP-H 0.00E+00 2.60E−08 94.1 MP-H2 2.60E−09 3.50E−08 35.6 MP-H3 4.70E−10 1.00E−08 71.2 MP-H4 0.00E+00 7.40E−09 63.6 MP-I 0.00E+00 1.60E−08 61 MP-I2 0.00E+00 6.40E−09 83.9 MP-I3 0.00E+00 1.00E−08 89 MP-J 0.00E+00 0.00E+00 223.7 MP-J2 0.00E+00 0.00E+00 142.4 MP-J3 0.00E+00 0.00E+00 137.3 MP3 6.40E−08 9.70E−07 31.2 MP-K7 2.40E−09 3.30E−07 9.4 MP4 1.00E−09 1.60E−06 22.9 MP-K9 5.70E−09 3.30E−07 23.4 MP-K10 4.10E−11 1.10E−07 80.6 MP-K11 3.60E−10 6.80E−08 63.6 MP-K12 9.80E−10 5.40E−08 86.4 MP-K13 0.00E+00 6.70E−08 63.6 MP-K14 4.10E−09 0.00E+00 0.8 MP-L 0.00E+00 0.00E+00 129.7 MP-L2 0.00E+00 0.00E+00 93.8 MP-P 1.90E−09 1.40E−08 144.9 MP-P3 4.20E−09 9.20E−07 33.3 MP-P4 1.70E−08 5.70E−07 81.4 MP-P5 1.00E−09 2.20E−06 26.7 MP-P6 1.80E−09 4.20E−07 10.7 MP-P7 1.30E−09 2.80E−07 83.9 MP-P8 2.00E−09 4.80E−07 32.8 MP-P9 1.20E−07 2.60E−05 2.9 MP6 9.00E−09 2.30E−05 1.7 MP-P11 6.10E−09 1.90E−05 1.4 MP-Q 1.60E−09 4.90E−07 14.7 MP-Q2 5.70E−09 3.40E−07 20.6 MP-Q3 8.70E−10 3.40E−07 16.8 MP-Q4 7.10E−09 9.70E−07 15 MP-Q5 7.00E−09 4.00E−07 22.5 MP-Q6 3.60E−09 6.70E−07 16.3 MP5 1.80E−08 7.40E−06 3.8 MP-Q8 6.20E−10 1.80E−07 37.4 MP-Q9 9.20E−10 6.90E−08 73.7 MP-Q10 8.30E−09 3.80E−08 81.4 MP-Q11 4.30E−10 4.20E−08 66.1 MP-Q12 8.80E−10 2.00E−08 144.9 MP-Q13 1.50E−09 1.60E−08 101.7 MP-R 3.50E−09 6.00E−07 13.9 MP-R2 2.80E−08 8.70E−07 13.1 MP-R3 4.10E−08 4.30E−06 4.3 MP-R4 8.60E−09 2.50E−06 3.7 MP-R5 1.80E−07 1.60E−05 5.8 MP-R6 2.10E−08 6.30E−05 0.7 MP-S 1.20E−10 2.30E−08 115.3 MP-S2 6.30E−11 2.10E−08 313 MP-S3 2.50E−10 9.80E−08 428.4 MP-T 7.00E−10 1.00E−06 13.6 MP-T2 7.10E−09 3.50E−07 23.1 MP-T3 3.20E−08 5.70E−07 12.6 MP-T4 2.20E−08 8.90E−07 13.7 MP-T5 2.30E−08 6.00E−07 21.7 MP-T6 9.20E−09 8.40E−07 16 MP-U 7.00E−09 9.80E−05 0.6 MP-U2 2.90E−09 1.10E−05 1.7 MP-U3 2.40E−08 5.50E−06 2.8 MP-U4 5.00E−09 9.20E−06 3.7 MP-V 1.70E−09 5.40E−07 5.7 MP-V2 2.90E−09 1.10E−06 7.9 MP-V3 1.10E−08 2.40E−06 3.4 MP-V4 5.00E−09 2.20E−06 4.6 MP-V5 8.70E−10 1.90E−06 6.2

Cytosine Deamination and Reduced Base Excision Repair

Overexpression of the catalytic domains of several cytidine deaminases in E. coli has been shown to have a mutagenic effect, resulting in primarily C→T transitions through a deoxyuracil intermediate [22]. The cytidine deaminase CDA 1 from Petromyzon marinus is reported to mediate the efficient mutation of prokaryotic and eukaryotic genomic DNA [22]. Additional mutations or deletion of the E. coli uracil-DNA glycosylase ung synergize with the effect of the deaminase and can enhance mutagenesis by disruption of the native uracil-excision repair pathway [22] (FIG. 7). Two natural protein inhibitors of Ung, Ugi and p56 [23], inhibit Ung through mimicry of structural and electronic features of uracil-containing DNA [23]. We hypothesized that the inclusion of a cytidine deaminase and uracil-DNA glycosylase inhibitor would further increase the potency of the MP through impairment of the uracil-excision repair pathway.

We placed ugi and cda1 downstream of dnaQ926, dam and seqA to yield MP5, and observed a 5-fold mutagenesis potency increase under induced conditions, representing an 18-fold increase in background mutagenesis compared to MP4 (FIG. 1A). This background increase was caused by two predicted σ70 promoters at the 3′ end of the seqA open reading frame, resulting in constitutive ugi and cda1 transcription (FIG. 9). We considered this background mutation rate for MP5 to be acceptable, as it was only ˜5-fold higher than the starting MP1. Alternative cytosine deaminases, including rat APOBEC1 and human AID, generally resulted in weaker effects on mutation rate than CDA1, in agreement with previous reports (CDA1>>AID≈APOBEC1) [22] (Tables 2 and 3). Overall, MP5 yielded 2.0×10−6 substitutions per bp per generation, a 31-fold increase in mutation rate relative to MP1 (Tables 2 and 3).

Impairing Mutagenic Nucleobase Export

Two major determinants of base selection during DNA replication are the catalytic alpha subunit of E. coli DNA Pol III, and regulation of the intracellular pools of dNTPs available during replication (FIG. 7). Mutations affecting the former are generally not viable or exert a mutator effect through reduced affinity to the proofreading domain, DnaQ [24], whereas perturbations affecting the latter are generally more tolerated and can be modified to affect the mutational spectrum [25]. We screened a number of mutator proteins that are known to compromise intracellular dNTP pools, and found overexpression of the emrR transcriptional repressor to be the most promising (FIG. 1A; Tables 2 and 3).

TABLE 4 Comparison of MP1-MP6 with previously described mutator plasmids. In each case, the mutator genes are listed with the source organism(s): Ee = Escherichia coli; Sc = Saccharomyces cerevisiae; Hs = Homo sapiens; Rn = Rattus norvegicus; Pa = Pseudomonas aeruginosa; Ll = Lactococcus lactis; PBS2 = Bacillus subtilis phage PBS2; Pm = Petromyzon marinus. In all cases, the fraction of cells showing rifampin resistance (RifR) was used to calculate μbp as described in the methods section, using R = 21 sites, N = 108, and N0 = 1.5 × 107 to approximate the levels as compared to the MP1-6 series. The fold increase in mutagenesis is shown for each MP (defined as the ratio of the mutagenesis in the strain without the MP vs. in the strain with the MP). All MPs are compared to MP6 in total mutagenesis efficiancy. *In cases where the MP was inducible, the dynamic range represents the fold increase between the uninduced and induced states for strains carrying the MP. Source Fraction RifR μbp (bp−1 generation−1) vs MP6 Dynamic organism Gene(s) −MP +MP −MP +MP Fold (%) range* Ref. Ec Dam 8.10E+08 3.00E−07 2.035+07 7.53E−09 0 0 — 48 Ec dnaE173 5.00E−09 7.20E−06 1.26E−10 1.81E−07 1440 1 24 24 Ec dnaQ926 3.00E−08 3.47E−04 7.53E−10 8.71E−06 11567 38 — 40 Ec mutD5 3.00E−08 5.70E−05 7.53E−10 1.43E−06 1900 6 — 40 Sc mag1 5.13E−08 3.61E−06 1.29E−09 9.06E−08 70 0 — 49 Sc mag1 1.00E−08 2.00E−06 2.51E−10 5.02E−08 200 0 200 54 Ec dinB 4.00E−08 4.55E−06 1.00E−09 1.14E−07 114 0 114 55 Ec dnaE (K655Y) 1.00E−08 8.45E−06 2.51E−10 2.12E−07 845 1 — 42 Hs AID 1.30E−08 1.03E−07 3.26E−10 2.59E−09 8 0 8 56 Rn APOBEC1 2.53E−08 1.23E−05 6.36E−10 3.09E−07 486 1 14 57 Rn APOBEC2 2.53E−08 2.50E−08 6.36E−10 6.28E−10 1 0 — 57 Hs AID 2.53E−08 1.66E−07 6.36E−10 4.17E−09 7 0 — 57 Hs APOBEC3C 2.53E−08 2.93E−07 6.36E−10 7.35E−09 12 0 — 57 Hs APOBEC3G 2.53E−08 2.70E−07 6.36E−10 6.78E−09 11 0 — 57 Ec mutH (E56A) 4.40E−08 8.74E−06 1.10E−09 2.19E−07 199 1 — 21 Ec mutH (K116E) 4.40E−08 8.40E−06 1.10E−09 2.11E−07 191 1 — 21 Ec mutH (K79E) 4.40E−08 7.00E−06 1.10E−09 1.76E−07 159 1 — 21 Ec mutH (B77A) 4.40E−08 6.94E−06 1.10E−09 1.74E−07 158 1 — 21 Ec mutH (D70A) 4.40E−08 6.88E−06 1.10E−09 1.73E−07 156 1 — 21 Ec mutH CΔ5 4.40E−08 9.10E−07 1.10E−09 2.28E−08 21 0 — 21 Ec mutL (R95F/N302A) 4.40E−08 1.17E−05 1.10E−09 2.94E−07 267 1 — 21 Ec mutL (R261H) 4.40E−08 1.04E−05 1.10E−09 2.62E−07 237 1 — 21 Ec mutL (E29A) 4.40E−08 8.32E−06 1.10E−09 2.09E−07 189 1 — 21 Ec mutL (K307A) 4.40E−08 6.75E−06 1.10E−09 1.69E−07 153 1 — 21 Ec mutL (N302A) 4.40E−08 3.50E−06 1.10E−09 8.79E−08 80 0 — 21 Ec mutL (R95F) 4.40E−08 1.27E−07 1.10E−09 3.19E−09 3 0 — 21 Ec mutL (K159E) 4.40E−08 7.26E−06 1.10E−09 1.82E−07 165 1 — 21 Ec mutL (R266E) 4.40E−08 6.05E−06 1.10E−09 1.52E−07 138 1 — 21 Ec mutL (R177E) 4.40E−08 1.77E−06 1.10E−09 4.44E−08 40 0 — 21 Ec mutL (I90R) 4.40E−08 1.56E−07 1.10E−09 3.92E−09 4 0 — 21 Ec mutL (R237E) 4.40E−08 1.40E−07 1.10E−09 3.51E−09 3 0 — 21 Ec mutL (G238A) 4.40E−08 5.40E−08 1.10E−09 1.36E−09 1 0 — 21 Ec mutL (G238D) 4.40E−08 1.03E−05 1.10E−09 2.59E−07 234 1 — 21 Ec mutL (I90E) 4.40E−08 1.70E−08 1.10E−09 4.27E−10 0 0 — 21 Ec mutS (S668A/T669V) 4.40E−08 1.33E−05 1.10E−09 3.34E−07 303 1 — 21 Ec mutS (K620M) 4.40E−08 1.21E−05 1.10E−09 3.05E−07 276 1 — 21 Ec mutS (D693A) 4.40E−08 1.15E−05 1.10E−09 2.90E−07 262 1 — 21 Ec mutS (E694Q) 4.40E−08 1.13E−05 1.10E−09 2.84E−07 257 1 — 21 Ec mutS (E694A) 4.40E−08 8.84E−06 1.10E−09 2.22E−07 201 1 — 21 Ec mutS (E694A/S668A/T669V) 4.40E−08 8.02E−06 1.10E−09 2.01E−07 182 1 — 21 Ec mutS (D693N) 4.40E−08 5.58E−06 1.10E−09 1.40E−07 127 1 — 21 Ec mutS (H760A) 4.40E−08 8.01E−07 1.10E−09 2.01E−08 18 0 — 21 Ec mutS (H728A) 4.40E−08 4.48E−07 1.10E−09 1.12E−08 10 0 — 21 Ec mutS (F596A) 4.40E−08 3.14E−07 1.10E−09 7.88E−09 7 0 — 21 Ec mutS (S612A) 4.40E−08 3.70E−08 1.10E−09 9.29E−10 1 0 — 21 Ec mutS (F36A) 4.40E−08 1.06E−05 1.10E−09 2.66E−07 241 1 — 21 Ec mutS (E38Q) 4.40E−08 4.19E−06 1.10E−09 1.05E−07 95 0 — 21 Ec mutS (D162R/E164R) 4.40E−08 4.30E−06 1.10E−09 1.08E−07 98 0 — 21 Ec mutS (R163E) 4.40E−08 3.90E−08 1.10E−09 9.79E−10 1 0 — 21 Ec mutS (R197E/R193E) 4.40E−08 1.11E−05 1.10E−09 2.79E−07 253 1 — 21 Ec mutS (R197E/R198E/R199E) 4.40E−08 6.15E−06 1.10E−09 1.54E−07 140 1 — 21 Ec mutS (E177A) 4.40E−08 1.10E−05 1.10E−09 2.75E−07 249 1 — 21 Ec mutS (T115A) 4.40E−08 7.99E−06 1.10E−09 2.01E−07 132 1 — 21 Ec Dam 3.00E−08 2.00E−06 7.53E−10 5.02E−08 67 0 — 17 Ec emrR 3.00E−08 5.00E−06 7.53E−10 1.26E−07 167 1 — 17 Ec mutS* 3.00E−08 3.00E−07 7.53E−10 7.53E−09 10 0 — 17 Ec seqA 3.00E−08 8.00E−07 7.53E−10 2.01E−08 27 0 — 17 Ec dinB 3.00E−08 3.00E−07 7.53E−10 7.53E−09 10 0 — 17 Pa nfx3 8.00E−08 3.50E−06 2.01E−08 8.79E−08 44 0 — 26 Ll dnaN 8.00E−08 9.80E−08 2.01E−09 2.46E−09 1 0 — 26 Ll dnaA 8.00E−08 1.90E−07 2.01E−09 4.77E−09 2 0 — 26 Ll uvrA, ysjE 8.00E−08 3.90E−08 2.01E−09 9.79E−10 0 0 — 26 Ll uvrA 8.00E−08 1.10E−07 2.01E−09 2.76E−09 1 0 — 26 Ll mhA, sipL, purR 8.00E−08 4.80E−08 2.01E−09 1.20E−09 1 0 — 26 Ll mhA, sipL 8.00E−08 6.70E−08 2.01E−09 1.68E−09 1 0 — 26 Ll nhA 8.00E−08 1.40E−08 2.01E−09 3.51E−10 0 0 — 26 Ec polB 2.20E−08 2.32E−06 5.52E−10 5.82E−08 105 0 116 52 Ec polB Q779V 2.20E−08 6.14E−08 5.52E−10 1.54E−09 3 0 — 52 Ec polB Δ780-783 2.20E−08 5.93E−08 5.52E−10 1.49E−09 3 0 — 52 Ec polBB (D156A) 2.20E−08 4.73E−04 5.52E−10 1.19E−05 21478 52 — 52 Ec uvrAB 7.90E−09 5.40E−08 1.98E−10 1.36E−09 7 0 — 58 Ec uvrABC 7.90E−09 1.90E−07 1.98E−10 4.77E−09 24 0 — 58 Hs AID 1.00E−08 1.40E−07 2.51E−10 3.51E−09 14 0 — 51 Hs AID (K10E/E156G) 1.00E−08 6.30E−07 2.51E−10 1.58E−08 63 0 — 51 Hs AID (K34E/K160E) 1.00E−08 4.20E−07 2.51E−10 1.05E−08 42 0 — 51 Hs AID 2.40E−08 4.90E−07 6.02E−10 1.23E−08 20 0 — 59 Hs AID-3FL 2.40E−08 3.70E−07 6.02E−10 9.29E−09 15 0 — 59 Hs AID-3GL 2.40E−08 1.60E−07 6.02E−10 4.02E−09 7 0 — 59 Hs AAG 1.00E−09 2.00E−07 2.51E−11 5.02E−09 200 0 1 50 Hs AAG (Y127I/H136L) 1.00E−09 1.15E−05 2.51E−11 2.89E−07 11500 1 4 50 Ec nrdAB 1.00E−08 8.00E−08 2.51E−10 2.01E−09 8 0 — 25 Ec nrdEF 1.00E−08 3.50E−07 2.51E−10 8.79E−09 35 0 — 25 Ec nrdA (H59A)B 1.00E−08 3.80E−07 2.51E−10 9.54E−09 38 0 — 25 Hs APOBEC3G 2.60E−07 2.40E−06 6.53E−09 6.02E−08 9 0 — 60 Hs APOBEC3G (E259Q) 2.60E−07 3.60E−07 6.53E−09 9.04E−09 1 0 — 60 Hs APOBEC3G (E254R) 2.60E−07 3.50E−06 6.53E−09 8.79E−08 13 0 — 60 Hs APOBEC3G (R313R) 2.60E−07 2.60E−07 6.53E−09 6.53E−09 1 0 — 60 Hs APOBEC3G (R320E) 2.60E−07 5.80E−07 6.53E−09 1.46E−08 2 0 — 60 Hs APOBEC3G (R313E/R320E) 2.60E−07 3.10E−07 6.53E−09 7.78E−09 1 0 — 60 Hs APOBEC3G (R374E) 2.60E−07 5.50E−07 6.53E−09 1.38E−08 2 0 — 60 Hs APOBEC3G (R376E) 2.60E−07 1.90E−06 6.53E−09 4.77E−08 7 0 — 60 Hs APOBEC3G (R374E/R376E) 2.60E−07 6.90E−07 6.53E−09 1.73E−08 3 0 — 60 Hs APOBEC3G (R213E) 2.60E−07 4.40E−07 6.53E−09 1.10E−08 2 0 — 60 Hs APOBEC3G (R215E) 2.60E−07 3.20E−07 6.53E−09 8.03E−09 1 0 — 60 Hs APOSEC3G (R213E/215E) 2.60E−07 2.90E−07 6.53E−09 7.28E−09 1 0 — 60 Ec dnaQ926, umuD′, umuC, recA730 7.35E−09 2.15E−05 7.10E−11 2.36E−07 3320 1 78 12 Ec dnaQ926 7.35E−09 2.98E−05 7.10E−11 3.61E−07 5087 2 355 This work Ec dnaQ926, dam 7.35E−09 5.54E−05 7.10E−11 9.74E−07 13723 4 15 This work Ec dnaQ926, dam, seqA 7.35E−09 1.03E−04 7.10E−11 1.60E−06 22610 7 1604 This work Ec, PBS2, dnaQ926, dam, seqA, ugi, cda1 7.35E−09 2.88E−04 7.10E−11 7.36E−06 103749 32 419 This work Pm Ec, PBS2, dnaQ926, dam, seqA, emrR, ugi, cda1 7.35E−09 4.73E−04 7.10E−11 2.29E−05 322414 100 34941 This work Pm

Derepression by EmrR results in upregulation of emrAB, which produces a multidrug efflux pump responsible for antibiotic resistance and the putative export of mutagenic nucleobase intermediates [26]. In an unbiased screen, emrR overexpression was found to have a potent mutator effect [17], presumably as a consequence of retaining these mutagenic nucleobases within the cell. To decrease background mutagenesis compared to MP5, we placed the emrR cassette between seqA and ugi, thereby disrupting the strong predicted σ70 promoter, yielding MP6. MP6 exhibited 2-fold lower background mutagenesis, while improving the overall mutator effect under induced conditions by 3-fold (FIG. 1A).

Features of the MP6 Mutagenesis System and Comparison with Other Methods

We chose MP6 for in-depth characterization because it offered the highest mutagenic potency with acceptable levels of toxicity (>1% of cells surviving under maximal MP induction) under the tested conditions. Efforts to increase the expression level of the six key genes in MP6 resulted in substantially higher toxicity or reduced potency (Tables 2 and 3). When induced, MP6 results in a 322.000-fold increase in mutation rate of chromosomal DNA over that of wild-type E. coli, and a 100-fold increase in mutation rate relative to that of MP1. Induced MP6 results in an average of 2.3×10−5 substitutions/bp/generation, representing to our knowledge the most potent inducible and genetically encodable mutagenesis method in bacteria reported to date (Table 4). This potency compares favorably to overexpression of PolB (D156A) (μbp=3.2×10−6), dnaQ926 (μbp=2.4×10−6), or mutD5 (μbp=3.9×10−7) (Table 4). We note that most previously published mutagenic constructs suffer from a lack of inducibility, have deleterious effects on host viability, and require overexpression of the mutagenic protein. In contrast, the MPs described here rely on the low-level expression of multiple genes, thereby affecting multiple pathways and enabling broad mutagenic spectra.

Additional permutations of this design or the inclusion of alternative mutators impaired overall mutation rate, or strongly decreased host viability, as evidenced by the characteristics of 80 candidate MPs with mutation rates spanning five orders of magnitude (FIGS. 10, 11, and 12; Tables 2 and 3). We observed that a loss of bacterial viability proportional to mutagenic potency beyond a mutation rate of ˜4×10−7 substitutions per bp per generation, corresponding to an average of ˜1.9 substitutions/genome/generation. Given that −10-30% of the E. coli genome has been estimated to be essential [27], this mutation rate threshold corresponds to ˜0.2-0.6 mutation in an essential gene/generation.

Next we tested the dynamic range and inducibility of MP6. To limit background mutagenesis, we increased the concentration of glucose from 25 mM to 200 mM during the transformation and growth stages prior to induction to maximize catabolite repression of the arabinose-inducible promoter. Under these modified conditions, induction of log-phase cultures of MG1655 ΔrecA::apra carrying MP6 with increasing concentrations of arabinose revealed a 35,000-fold dynamic range between 10 μM and 100 mM arabinose (FIG. 2A). Despite this very strong induction effect, MP6-carrying cultures maintained low levels of mutagenesis when suppressed with 200 mM glucose (FIG. 2A).

To further evaluate MP6, we compared its performance with that of the most commonly used in vivo mutagenesis strain, XL1-Red (Agilent Technologies). XL1-Red is deficient in proofreading (mutD5), mismatch-repair (mutS) and base-excision (mutT) activities, resulting in high levels of substitutions in chromosomal and episomal DNA [6]. However, the strain grows very slowly, is difficult to manipulate genetically, has poor transformation efficiency, and produces a fairly narrow mutagenic spectrum [7]. Using the rifampin resistance assay, we found that XL1-Red results in 29 substitutions/genome/generation, while MP6 in the induced state produces an average of 110 substitutions/genome/generation, a ˜4-fold higher mutation rate (FIGS. 2B, 2C). The uninduced state of MP6 yields similar background mutagenesis levels (0.01 substitutions/genome/generation) as the non-mutagenic related strain XL1-Blue (0.005 substitutions/genome/generation) (FIGS. 1A, 1B, 2C). Together, these results establish that MP6 allows for mutagenesis levels exceeding that of the most commonly used mutator E. coli strain, and offers the ability to control mutation rate with an exogenous small molecule.

MP6 Augments M13 Bacteriophage Mutagenesis

To further characterize MP1, MP4, and MP6, we assessed their impact on the mutagenesis of bacteriophage DNA. Phage display is a widely used platform for laboratory screening and evolution efforts, and has been used to generate many proteins with novel or improved activities [28]. We measured the mutagenesis rate of M13 phage in host cells harboring a variety of MP variants or in XL1-Red using a lacZ inactivation assay. Briefly, a lacZ cassette was integrated downstream of geneIII in the wild-type M13 genome to yield SP063, resulting in high-level expression of β-galactosidase and the production of blue plaques in the presence of an X-Gal analog (FIG. 13). Disruption of the lacZ cassette due to high mutagenesis reduces or ablates this blue-plaque phenotype, enabling the estimation of phage mutagenesis rates. We compared the ratio of white:blue plaques (lacZ phenotype) using MP1, MP4, MP6, and XL1-Red.

Under conditions supporting phage inoculum expansion in overnight culture using S1030 cells (FIG. 14 and Table 1), we observed that the phage-borne lacZ inactivation rates scaled with MP potency, reaching up to 27% white plaques (representing mutant, inactive lacZ genes) after 18 to 24 h of culture with MP6 (FIG. 3A). As MP6 increases the rate of mutagenesis by ˜100-fold as compared to MP1, the expected M13 bacteriophage mutation rate is elevated to 7.2×10−3 substitutions/bp/generation [11], resulting in an average of 22 substitutions/genome/generation in the SP063 phage. This mutagenesis potency of 2.3 substitutions/kb (0.23%) approaches the potency of the most commonly used in vitro mutagenesis methods, Mutazyme II (0.3%-1.6%, Agilent Technologies).

To allow for a comparison to XL1-Red, which lacks an F′ episome and thus cannot be infected by M13 phage particles but allows for the production of fully functional phage, purified SP063 dsDNA was transformed into S1021 (the F− variant of S1030, Table 1) cells carrying the aforementioned MPs (induced prior to or after SP063 transformation, or both; FIG. 15) or XL1-Red. Whereas phage produced from transformed XL1-Red cells only yielded an average of 5% white plaques on S1030 cells (FIG. 3B), phage produced from the S1021 strain carrying MP6 yielded 15% white plaques and MP1 and MP4 yielded 7% and 10% white plaques, respectively, on S1030 cells (FIG. 3C). These results further demonstrate the greater mutational potency of MP6 compared to that of XL1-Red, and also highlight the strain flexibility achieved by using inducible, genetically encodable mutagenesis systems.

Mutational Spectra of Designed MPs

In addition to mutagenic potency, the distribution of mutation types is also important, as a narrow mutational spectrum limits the diversity of changes that can be accessed. To analyze the spectrum of produced mutations, we took advantage of previously reported distributions of rifampin-resistant rpoB mutants. Importantly, mutations covering each of the 12 possible transitions and transversions in the rpoB gene are known to endow E. coli with resistance to high levels of rifampin [29]. We assessed the spectra of MP1, MP4, and MP6 by sequencing rifampin-resistant rpoB alleles in mutated MG1655 ΔrecA::apra, and compared the spectra of each to the rpoB mutation spectrum afforded by XL1-Red (FIGS. 4A-D). MP1 yielded a narrow mutagenic spectrum strongly biased towards A:T→T:A transversions, a known side-effect of using recA730-based mutators on genomic DNA [30]. Comparatively, the intermediate MP4 had a more uniform distribution, covering more transitions and a marked increase in G:C→A:T and A:T→G:C, a hallmark of mutagenesis methods that perturb the mismatch repair response [31]. MP6 exhibited a wider spectrum still, with a more uniform distribution of transitions and transversions. The identities of the observed rifampin-resistant mutations are in agreement with previous studies (FIG. 4E) [29]. Notably, XL1-Red exclusively displayed two types of mutations, C:G→T:A and T:A→C:G transitions (FIGS. 4D, 4E). This observation is consistent with previous reports describing the narrow mutational spectrum of XL1-Red [7]. Additional MP characterization using the established β-galactosidase (lacZ) reversion strains developed by Miller and coworkers [32] revealed similar mutational spectra as observed in the rifampin resistance assays (FIG. 16).

To further characterize the mutagenic spectra, we propagated the lacZ-encoding phage SP063 using 51030 strains carrying MP1, MP4 and MP6, or transformed SP063 DNA directly into XL1-Red to produce progeny phage, and subjected the lacZ ORF from all the resultant phage to high-throughput DNA sequencing (FIGS. 4F-I). The mutagenesis efficiency for all conditions was in agreement with that revealed by the other assays (FIGS. 4A-I, 16). The MPs generated broad mutagenic spectra in progeny phage, consistent with both rpoB single clone sequencing and lacZ reversion assays, with the exception of MP1, which showed a more uniform distribution of mutation types using the phage assay and lacZ reversion assays than using rpoB sequencing. This discrepancy is likely a result of the MP1-encoded recA730 allele, which selectively enhances the rate of A:T→T:A transversions in DNA of strains lacking recA (MG1655 ΔrecA::apra), as it no longer competes for substrates with wild-type (CSH strains [32], Table 1) or reduced-activity recA mutants (S1021 and S1030, Table 1) for function. Phage sequences produced from XL1-Red revealed a much narrower mutational spectrum, with a bias for A:T→C:G mutations. Taken together, these results reveal that the MPs developed in this study can outperform the most widely used in vivo and in vitro mutagenesis techniques (FIG. 17) both in mutagenic potency and mutational breadth.

Evolution of Antibiotic Resistance Using the Designed MPs

To evaluate the impact of these MPs on laboratory evolutionary outcomes, we evolved antibiotic resistance in E. coli, as well as RNA polymerase substrate specificity changes in bacteriophage, using cells harboring different MPs. First we tested the ability of MP1, MP4, or MP6 to evolve resistance of E. coli strain MG1655 ΔrecA::apra to a number of commonly used antibiotics. Mid-log-phase cultures of MG1655 ΔrecA::apra carrying MP1, MP4, or MP6 were induced for 18-21 h, then serially diluted and plated on agar plates without antibiotics, or with 5-100 μg/mL of carbenicillin, cephotaxime, fosfomycin, kanamycin, metronidazole, norfloxacin, rifampin, spectinomycin, streptomycin, or tetracycline. The antibiotic concentrations used in all ten cases were well above known MIC values (Table 5).

TABLE 5 Minimum inhibitory concentrations (MICs) for selected antibiotics. All data regarding E. coli MICs was tabulated from the Antimicrobial Index Knowledgebase (antibiotics.toku-e.com). Antibiotic MIC (ug/mL) carbenicillin  2-25 cefotaxime 0.016-0.25  fosfomycin 0.125-8     kanamycin 0.25-8    metronidazole  8-32 norfloxacin 0.016-0.125 rifampin 0.5-16  spectinomycin  8-64 streptomycin  1-16 tetracycline 0.5-8  

After only 18 hours of growth on antibiotic-containing solid medium, large fractions of the bacterial population showed resistance to high concentrations of carbenicillin, fosfomycin, kanamycin, metronidazole, rifampin, spectinomycin, streptomycin, and tetracycline (FIG. 5A and Table 6). No resistant colonies were detected for cefotaxime (a third-generation cephalosporin) or norfloxacin (a synthetic fluoroquinolone). The frequency of antibiotic resistance strongly correlated with MP potency (FIG. 5A). For example, we observed the evolution of resistance to high levels of kanamycin with no intermediate selection step using MP6, which allowed for up to 11% of the total population to grow in the presence of 30 μg/mL kanamycin. In contrast, only 0.02% or 0.66% of the population survived this level of kanamycin when using MP1 or MP4, respectively (FIG. 5A).

TABLE 6 Comparison of developed MPs to chemical mutagens, UV light and XL1-Red. The fraction of cells resistant to each antibiotic upon mutagenic treatment is shown for all tested antibiotics. No resistance was observed for norfloxacin using any MP, chemical mutagen, or strain. All MPs, chemical mutagens, and UV light treatments used E. coli MG1655 ΔrecA::apra. CRB, 50 μg/mL carbenicillin; CTX, 5μg/mL cefotaxime; FOS, 100 μg/mL fosfomycin; KAN, 30 μg/mL kanamycin, MTX, 100 μg/mL metronidazole; RIF, 100 μg/mL rifampin; SPC, 100 μg/mL spectinomycin; STR, 50 μg/mL streptomycin; TET, 10 μg/mL tetracycline. We note that MP1 (recA730), XL1-Blue (recA1), and XL1-Red (wt recA) are all proficient at recombination, a known requirement for high-level resistance to metronidazole61. Additionally, XL1-Blue and XL1-Red are both inherently resistant to tetracycline, explaining the observed high incidence of resistance. CRB CTX FOS KAN MIX RIF SPC STR TET no MP 0.00E+00 0.00E+00 1.10E−05 5.78E−06 2.22E−07 5.56E−08 0.00E+00 6.67E−07 0.00E+00 MP1 9.57E−06 0.00E+00 9.17E−04 1.84E−04 6.47E−01 1.26E−05 1.91E−07 5.68E−06 0.00E+00 MP4 1.37E−05 0.00E+00 6.97E−03 6.56E−03 1.52E−06 6.17E−05 1.14E−05 1.56E−05 5.97E−06 MP6 7.39E−04 0.00E+00 2.78E−02 1.11E−01 1.50E−04 7.17E−04 2.55E−04 6.22E−04 1.15E−04 2AP 1.17E−07 2.80E−06 4.83E−04 1.53E−03 2.03E−05 1.03E−05 2.50E−07 3.17E−07 0.00E+00 EMS 0.00E+00 1.67E−07 3.75E−04 8.17E−04 1.10E−04 6.68E−05 0.00E+00 1.33E−06 0.00E+00 MNNG 0.00E+00 3.67E−07 1.22E−04 5.33E−04 3.22E−05 5.56E−06 3.67E−05 2.44E−07 0.00E+00 UV 0.00E+00 0.00E+00 5.00E−07 7.49E−06 0.00E+00 0.00E+00 0.00E+00 2.89E−08 0.00E+00 XL1-Blue 0.00E+00 5.40E−06 0.00E+00 8.66E−07 4.82E−01 0.00E+00 0.00E+00 0.00E+00 9.44E−01 XL1-Red 0.00E+00 0.00E-00 1.42E−03 2.62E−04 9.84E−01 3.62E−05 3.81E−06 3.60E−05 1.10E−04

We repeated these antibiotic resistance evolution experiments to compare the performance of these MPs to those of the commonly used chemical mutagens [32] ethyl methanesulfonate (EMS), methylnitronitrosoguanidine (MNNG), and 2-aminopurine (2AP), as well as UV irradiation, XL1-Red, and XL1-Blue (FIGS. 5A, 5B: Table 6). MP6 outperformed all six of the other mutagenesis treatments or strains for all but one of the antibiotics. Cephotaxime resistance was not observed from use of any of the MPs, but was weakly detected from the chemical mutagens (FIG. 6B). We speculate that the known ability of all three of these chemical mutagens to greatly enhance G:C→A:T transitions may contribute to the evolution of cephotaxime resistance. Cumulatively, these results suggest that MP6 can rapidly generate strains with novel properties, outperforming several commonly using chemical mutagens, UV irradiation, and XL-1 Red.

MP6 Allows for Direct RNA Polymerase Evolution During PACE

Next we compared the performance of MP1 and MP6 during phage-assisted continuous evolution (PACE) [11, 12, 33-35] of T7 RNA polymerase. We previously showed that PACE can evolve T7 RNA polymerase to recognize the distant T3 promoter (PT3) (FIG. 18), but only using either an intermediate evolutionary stepping-stone (a T7/T3 hybrid promoter) [11, 33, 34], or an initial period of evolutionary drift in the absence of selection pressure [12]. Without either an evolutionary stepping-stone or initial evolutionary drift, PACE cannot evolve T7 RNAP variants that recognize PT3 [11, 12, 33, 34]. We hypothesized that with improved mutagenesis, evolved solutions could be accessed before the phage population washed out without requiring evolutionary stepping-stones or modulated selection stringency.

We compared the ability of MP1 and MP6 to rapidly evolve PT3-active T7 RNAP variants in the absence of evolutionary drift, or under conditions in which the selection stringency was slightly reduced. Importantly, previous attempts of T7 RNAP evolution towards PT3 activity using MP1 required a drift period of virtually no selection pressure over 18 h to yield PT3-active variants, whereas the high or intermediate selection pressures resulted in rapid phage washout 1121.

In agreement with our previous results, T7 RNAP phage added to lagoons fed by host cells harboring MP1 under high or intermediate selection stringency rapidly washed out in <20 h of PACE when required to recognize PT3 (FIG. 6A). In contrast, both lagoons tested in which host cells harbored MP6 allowed for the propagation of T7 RNAP phage on host cells requiring Pm recognition under high or intermediate selection stringency, with nearly 10% of both populations after only 10 h of PACE exhibiting activity on PT3 by activity-dependent plaque assays (FIG. 6B). Sequencing confirmed mutations M219K/E222K/E222Q together with N748D in all surviving clones (FIG. 19), in agreement with previous findings of our group and others [11, 12, 33, 34, 36]. The ability of MP6 to support the discovery of P3-active T7 RNAP variants highlights the ability of this MP to access mutations more efficiently, and thus mediate a more thorough and larger sampling of sequence space. Collectively, these results establish that the enhanced mutagenesis mediated by the new MPs can support accelerated access to evolved proteins that are difficult to access directly using previous methods.

Discussion

Using a systematic, mechanism-guided approach, we developed a series of vectors that express a variety of genes known to adversely affect DNA replication fidelity. In total we generated and assayed 80 candidate MPs with mutation rates spanning five orders of magnitude (FIGS. 11 and 12; Tables 2 and 3). The resulting MPs support highly potent, broad-spectrum, inducible, vector-based in vivo mutagenesis that rival the performance characteristics of popular in vitro methods such as error-prone PCR, while offering key advantages of in vivo mutagenesis. These advantages include enabling mutation and selection cycles to be coupled, and bypassing transformation efficiency bottlenecks that limit the size of populations that can be generated from DNA diversified in vitro. The MPs developed here offer major advantages over current in vivo mutagenesis methods such as chemical mutagens or base analogs, UV irradiation, or constitutive hypermutator strains. Hypermutator strains, for example, generally suffer from poor transformation efficiency (XL1-Red, ˜1×106 cfu/μg plasmid DNA), high instability, and narrow mutagenic spectra. MP6 increases the mutation rate of E. coli by 322,000-fold, and substantially exceeds both the mutation rate and the mutagenic spectra of XL1-Red. Importantly, MP6 can support approximately ˜2.3 substitutions/kb in a gene of interest using phage vectors in a single generation, with additional increases in mutagenesis efficiency concomitant with longer propagation times.

To demonstrate the utility of these vectors, we used a whole-genome mutagenesis approach to evolve high-level antibiotic resistance in E. coli. In the absence of any prior selection or mutagenesis step, MP6 rapidly mediates the evolution of antibiotic resistance to many commonly used antibiotics within 18 h. The efficiency and effectiveness of antibiotic resistance mediated by MP6 compares favorably with that of a number of potent chemical mutagens (2AP, EMS, MNNG), UV irradiation, and the hypermutator strain XL1-Red. In addition, we observed that MP6 supports the continuous evolution of 17 RNA polymerase variants capable of initiating transcription at the non-cognate T3 promoter in less than 10 h, without requiring evolutionary stepping-stones or an initial period of evolutionary drift. The MPs provided herein are broadly applicable to the use of in vivo mutagenesis to provide efficient access to rare solutions in sequence space that would otherwise be much more difficult, or impossible, to reach using current in vitro or in vivo methods. The properties of MP6, which include very high mutagenesis efficiency, broad mutational spectrum, small-molecule inducibility, and compatibility with a variety of bacterial strains, together represent a substantial advance in in vivo mutagenesis methodology for the laboratory evolution community.

Materials and Methods

General methods. All PCR reactions were performed using Pfu Turbo Cx polymerase (Agilent Technologies) or VeraSeq ULtra polymerase (Enzymatics). Water was purified using a MilliQ water purification system (Millipore, Billerica Mass.). All MPs were constructed using USER cloning (New England Biolabs). Native E. coli genes were amplified by PCR directly from genomic DNA, and non-bacterial genes were synthesized as bacterial codon-optimized gBlocks Gene Fragments (Integrated DNA Technologies). All DNA cloning was carried out using NEB Turbo cells (New England Biolabs).

General MP strain preparation. Mid log-phase (OD600=˜0.5-0.8) cells of the strain of interest grown in 2×YT (United States Biological) were transformed with the desired MP, and recovered for 45 min in Davis rich media12 to suppress MP induction. All transformations were plated on 2×YT in 1.8% agar (United States Biological) containing 40 μg/mL chloramphenicol (Sigma Aldrich), 10 μg/mL fluconazole (TCI America), 10 μg/mL amphotericin B (TCI America), 25 mM glucose (United States Biological) and grown for 12-18 h in a 37° C. incubator. Colonies transformed with the appropriate MP were picked the following day and grown in Davis rich media containing 40 μg/mL chloramphenicol, 10 μg/mL fluconazole, and 10 μg/mL amphotericin B for 12-18 h. Following overnight growth of the MP-carrying strains, cultures were diluted 1,000-fold into fresh Davis rich media containing 40 μg/mL chloramphenicol, 10 μg/mL fluconazole, and 10 μg/mL amphotericin B. The remainder of each experiment is described in each of the following sections.

Rifampin resistance assay. Upon reaching mid log-phase, cultures were induced with 25 mM arabinose (Davis rich media+arabinose) or suppressed with 25 mM glucose (Davis rich media only) and allowed to continue growth for an additional ˜18-24 h in a 37° C. shaker. The high arabinose concentration ensures sufficient induction of the plasmid-borne mutators MG1655 ΔrecA::apra despite arabinose catabolism by this strain upon glucose depletion. For XL1-Blue and XL1-Red strains, cultures were started directly from glycerol stocks according to the manufacturer's instructions and incubated for an identical amount of time as the MP-carrying strains. After overnight growth, cultures were serially diluted in 10-fold increments and plated on 2×YT-agar containing 10 μg/mL fluconazole, 10 μg/mL amphotericin B, and 100 mM glucose+/−100 μg/mL rifampin. After 18-24 h, the number of colonies on the glucose+/−rifampin plates was counted for each culture. The mutation efficiency induced by the MP (μbp, substitutions/bp/generation) was calculated using the equation: μbp=f/[R×ln(N/N0)], where f is the frequency of rifampin-resistant mutants (as compared to the glucose control), R is the number of sequenced sites yielding rifampin resistance (21 sites across both rpoB clusters in our experiments), N is the final population size, and N0 is the population size at which resistance is first observed (empirically determined to be ˜1.5×107). To calculate μG, μbp was multiplied by the genome size, which for MG1655 was 4.64×106 bp.

Episomal lacZ reversion assay. Upon reaching mid log-phase, the cultures were induced with 25 mM arabinose or suppressed with 25 mM glucose, and allowed to continue growth for an additional ˜18-24 h. After overnight growth, the cultures were centrifuged for 2 min at 10,000× rcf and resuspended in an equal volume of 10% glycerol. This procedure was carried out twice to remove trace glucose or other carbon sources from the supernatant prior to plating. Washed cells were serially diluted in 10-fold increments using 10% glycerol and plated on M9 minimal media agar supplemented with 5 mM MgSO4, 0.01% thiamine, 335 μg/mL Bluo-Gal (Life Technologies) and either 10 mg/mL glucose or 10 mg/mL lactose. The Bluo-Gal was added to ensure that survival on lactose was concomitant with lacZ reversion, and not purely due to extracellular lactose hydrolysis. After extended growth (˜24-36 h), the fraction of lactose-catabolizing colonies was calculated using the number of blue colonies on the lactose plates vs. the total number of colonies on the glucose plates.

Phage lacZ inactivation assay. Upon reaching mid log-phase, the cultures were induced with 25 mM arabinose or suppressed with 25 mM glucose, allowed to grow for an additional 0-2 h, then, in the case of strain S1030, infected with SP063 phage, and allowed to grow for an additional˜18-24 h. For S1021 and XL1-Red (Agilent Technologies), SP063 DNA was miniprepped from infected S1030 cells and electroporated into these strains as they both lack F′ episomes. For F cells, cultures were either induced for 2 h prior to being made electrocompotent, induced immediately following transformation, induced both prior to and following electroporation, or not induced at all. After overnight growth and phage propagation, the cultures were centrifuged 2 min at 10,000× ref and the supernatant was filtered through a 0.2 μm PVDF filter (Millipore). The supernatant was serially diluted in 10-fold increments using Davis rich media and plaqued on S1030 cells using 1.8% 2×YT-agar for the bottom layer and 0.6% 2×YT-agar supplemented with 400 μg/mL Bluo-Gal (Life Technologies) for the top layer. The fraction of white or light blue plaques (lacZ− phenotype) was counted as a function of all plaques (blue+light blue+white), and used as a measure of mutation frequency for the lacZ cassette.

Sanger sequencing of rpoB mutations. Rifampin-resistant colonies were picked into 96 well plates and grown overnight in Davis rich media supplemented with 100 μg/mL rifampin. Following overnight growth, 10 μL aliquots were heated at 100° C. for 10 min, followed by PCR using primers AB1678 (5′-AATGTCAAATCCGTGGCGTGAC, SEQ ID NO: 20) and AB1682 (5′-TTCACCCGGATACATCTCGTCTTC, SEQ ID NO: 21) to amplify an rpoB fragment containing both clusters 1 and II. Each fragment was sequenced twice using primers AB1680 (5′-CGGAAGGCACCGTAAAAGACAT, SEQ ID NO: 22) and AB1683 (5′-CGTGTAGAGCGTGCGGTGAAA, SEQ ID NO: 23).

High-throughput sequencing of lacZ mutations. SP063 phage that was propagated using S1030 carrying MP1, MP4 or MP6, produced by XL1-Red following SP063 DNA electroporation, or the unmutated stock phage was amplified by PCR using primers AB437 (5′-GGCGCTGGTAAACCATATG, SEQ ID NO: 24) and DB213 (5′-GGAAACCGAGGAAACGCAA, SEQ ID NO: 25) to yield a ˜3,400 bp fragment containing the lacZ gene. SP063 phage that was propagated under similar conditions on S1030 cells was used as the negative control. Three biological replicates were carried out for each of the aforementioned samples. The resulting PCR products were purified by gel electrophoresis using a 1% agarose gel and prepared for HTS using a Nextera kit (Illumina) and a previously described procedure35. Briefly, 4 μL of DNA (2.5 ng/μL), 5 μL TD buffer, and 1 μL TDE1 were mixed together and then heated to 55° C. for 5 min. After purification (Zymo DNA purification kit), the resultant “tagmented” DNA samples were amplified with Illumina-supplied primers using the manufacturer's protocol. The resulting PCR products were then purified using AMPure XP beads and the final concentration of DNA was quantified using PicoGreen (Invitrogen) and qPCR. The samples were sequenced on a MiSeq Sequencer (illumina) in 2×300 paired-end runs using the manufacturer's reagents following the manufacturer's protocols.

High-throughput sequencing data analysis. A previously described custom MATLAB script35 (available upon request) was used to align MiSeq reads with Q score<30 to the wild-type sequence and count the nucleotide positions from which the experimental sample deviates from the wild-type sequence yielding called mutations with >99.9% accuracy, corresponding to >3 s.d, above the mean error rate of the MiSeq high-throughput sequencing reads. To compensate for systemic sample preparation and sequencing errors, the observed fraction of mutations at each nucleotide position of the wild-type lacZ reference gene was subtracted from the fraction of mutations in a given experimental sample to result in the “corrected fraction mutated”. Mutations were defined as nucleotide positions with a corrected fraction mutation that is both greater than the average corrected fraction mutated of the treatment of interest and at least one standard deviation higher than the corrected fraction mutation of the wild-type reference sequence. Duplicates belonging to set of paired-end reads were treated as a single sample, while duplicate reads of the same region with alternative adaptor/index sequences were not removed so as not to introduce bias into the sequencing analysis. This process yielded an average of ˜50,000 reads per position for each of the sequenced samples.

Evolution of novel antibiotic resistance. MG1655 ΔrecA::apra cells without an MP or carrying MP1, MP4, or MP6 were grown for 18-21 hr in Davis rich media containing 40 μg/mL chloramphenicol, 10 μg/mL fluconazole, 10 μg/mL Amphotericin B, and supplemented with 200 mM arabinose to induce the MPs. Small molecule and UV mutagenesis was carried out as previously described32. For 2AP treatment, log-phase MG1655 ΔrecA::apra cells were diluted to ˜1000 cells, the media was supplemented with 700 μg/mL 2AP (TCI America), and the culture was allowed for grow at 37° C. for an additional 18-21 hr. For EMS treatment, 2 mL of a log-phase MG1655 ΔrecA::apra culture (˜1×108-1×109 cells) was centrifuged, washed twice with 1 mL A buffer on ice, then supplemented with 14 μL EMS (TC America). Cells were lightly vortexed, and allowed to shake at 200 rpm at 37° C. for 45 min. After this time, the culture was centrifuged, washed twice with 1 mL A buffer on ice, diluted by 20-fold into Davis rich media without antibiotics, and allowed to grow for 18-21 hr. For MNNG treatment, 2 mL of a log-phase MG1655 ΔrecA::apra culture (˜1×108-1×109 cells) was centrifuged, washed twice with 1 mL citrate buffer (pH 5.5) on ice, supplemented with 111 μL of 1 mg/mL MNNG (TCI America) and placed in a 37° C. water bath for 30 min. Following treatment, the cells were centrifuged, washed twice with 1 mL 0.1 M potassium phosphate buffer (pH 7.0), diluted by 4-fold into Davis rich media without antibiotics, and allowed to grow for 18-21 hr. For UV irradiation, 2 mL of a log-phase MG1655 ΔrecA::apra culture (˜1×108-1×109 cells) was centrifuged, resuspended in 1 mL 0.1 M MgSO4 and placed on ice for 10 min. Cells were placed in a petri dish and exposed to UV light from a SM-36-2GR UV lamp (American Air & Water) for 1 min, uncovered, at a distance of ˜10 cm. Immediately following UV exposure, cells were diluted by 20-fold into Davis rich media without antibiotics, and allowed to grow for 18-21 hr. For XL1-Blue and XL1-Red strains, cultures were started directly from glycerol stocks according to the manufacturer's instructions and allowed to grow for 18-21 hr in Davis rich media. Following overnight growth, all cultures were serially diluted in Davis rich media and plated on 2×YT-agar containing 10 μg/mL fluconazole, 10 μg/mL amphotericin B, and 100 mM glucose+/−the appropriate antibiotic. After overnight growth (˜18-24 h), the numbers of colonies on the glucose+/−antibiotics plates were counted.

Continuous evolution of PT3-active T7 RNAP variants. Two modified versions of MP1 and MP6 (MP1a and MP6a, respectively) were generated to support robust phage propagation during PACE. These MPs carry all of the components of their respective MPs, in addition to the previously described anhydrotetracycline (ATc)-dependent drift promoter driving geneIII12. S1030 strains carrying either MP in addition to the PT3 accessory plasmid (AP) were inoculated into host-cell cultures (chemostats) and grown at a dilution rate of 1.6 vol/hr as previously described12. Lagoons flowing from the respective chemostats were maintained at 40 mL, diluted at 0.75 vol/hr, and supplemented with either 25 mM arabinose only (high stringency) or 25 mM arabinose with 30 ng/mL ATc (intermediate stringency) for 8 h prior to infection with packaged T7 RNAP SP. We note that concentrations exceeding 30 ng/mL for extended timeframes during PACE (>24 hr) result in excision of the evolving gene from the selection phage. As continuous flow conditions effectively enrich for SPs capable of rapid replication, selection phage with smaller genomes are rapidly enriched to totally dominate the evolving pool. Each lagoon was infected with 4×109 pfu, resulting in an initial titer of 108 pfu/mL of the lagoon. Samples were taken 10 h and 20 h after infection, centrifuged at 10,000 ref for 2 min, then sterile filtered with a 0.2 μm filter and stored overnight at 4° C. Phage aliquots were titered on S1030 cells carrying either the PSP-geneIII AP (total phage) or the PT3-geneIII AP (PT3-active phage).

MP6 optimization. We retained native bacterial ribosome-binding sites (RBSs) upstream of the ORFs for four of the six MP6 genes. The exceptions are dam, which natively lacks a canonical RBS in the E. coli genome, and cda1, which derives from the eukaryote P. marinus and thus does not use a bacterial Shine-Dalgarno sequence. In an attempt to further enhance mutational potency by modulating the expression of dnaQ926, dam, seqA, emrR, ugi, and cda1, we varied RBS upstream of each of these six genes by individually mutating them to fully complement the 16S rRNA, and resulting in optimal transcript translation. Interestingly, strengthening the RBSs upstream of each of the six genes generally reduced the potency of the MP, with the exception of the seqA and dnaQ926 RBSs (Tables 2 and 3). Strengthening the seqA RBS proved highly lethal under induced conditions, likely as a consequence of impeded genomic replication (Tables 2 and 3). Increasing the strength of the dnaQ926 RBS enhanced the mutagenic potency of the MP by 4-fold under induced conditions, concomitant with a minor increase in background mutagenesis, but was more toxic to bacteria as evidenced by a greater loss of viability under induced conditions (Tables 2 and 3). Additionally, measurement of the mutation rate of the resulting MP became irreproducible, consistent with MP instability under these conditions. These findings together suggest that additional mutagenic potency gains beyond that of MP6 may result in error catastrophe and reduced MP stability.

LacZ reversion analysis. Strains CSH101 to CSH106 each carry different nonfunctional missense mutants of lacZ at codon 461 (natively encoding glutamic acid) on the F′ episome. If a mutation reverts the nonfunctional codon to a glutamic acid, the strain can synthesize functional LacZ and survives using lactose as the only carbon source. These six strains are designed to report on all 12 possible mutations using this codon reversion. Using these strains, we observed that MP1 had the most narrow episomal mutational spectrum, with a moderate bias towards G:C→A:T (CSH102) and A:T→G:C (CSH106) substitutions (FIG. 17). MP4 showed an improved distribution of mutations, with a still moderate preference for A:T→G:C (CSH106) substitutions (FIG. 18). MP6 showed a near equal distribution of mutations in all six strains, with the exception of A:T→C:G (CSH101) substitutions which were detected at 10- to 100-fold lower levels than the other substitutions (FIG. 16). Taken together, these results suggest that MP6 generally outperforms the other MPs and XL1-Red based on both the frequency of lacZ reversion as well as the breadth of mutation types detected by these strains. Importantly, the mutational potency and spectra using the episomal lacZ reversion assays were in agreement with those from the rifampin resistance assays (FIGS. 4A-E, 17). These results establish the ability of the MPs to affect a wide variety of mutations in both genomic and episomal DNA.

Exemplary Mutagenesis Plasmid Sequence

The Sequence for an exemplary, non-limiting embodiment of a mutagenesis expression construct as provided herein, in this case a mutagenesis plasmid comprising a nucleic acid sequence encoding dnaQ926, dam, seqA, emrR, ugi, and CDA1 is provided below:

pAB086p10-MP CloDF13 pBAD dnaQ926 dam seqA emrR ugi CDA1 cat standard; circular DNA; 6681 BP. LOCUS pJC184 6537 bp DNA circular Key Location/Qualifiers terminator 867..911 /note=“rrnB1 transcriptional terminator” rep_origin complement(39..777) /dnas_title=“cloDF13” /vntifkey=“33” /label=cloDF13 terminator complement (5859..5894) /note=“P14/tonB bidirectional terminator” /note=“termination of cat transcript is slightly weaker than in opposite direction” CDS complement (5908..6567) /note=“cat (CmR)” /note=“from pACYCDuet-1” modified_base 5983..5983 /note=“mutation” /note=“annotated as a G in pACYCDuet cat marker annotation, here it is an A, this mutation is silent from codon GTC (Val, 25% codon usage) to GTT (Val, 21% usage)” promoter 6568..6665 /note=“cat promoter” /note=“from pACYCDuet−1” CDS complement (927..1805) /dnas_title=“araC” /vntifkey=“4” /label=araC misc_feature 1903..1903 /dnas_title=“C to A ***” /vntifkey=“21” /label=C to A *** misc_feature 1821..1821 /dnas_title=“A to G ***” /vntifkey=“21” /label=A to G *** prim_transcript complement (1969..1969) /note=“pC TSS” protein_bind 1992..2008 /note=“araO1” protein_bind 1971..1987 /note=“araO1” protein_bind 1834..1850 /note=“araO2” protein_bind 2013..2034 /note=“CAP” misc_feature 2093..2093 /note=“” misc_feature 2079..2079 /note=“” prim_transcript 2117..2117 /note=“pBAD TSS” protein_bind 2045..2061 /note=“araI1” protein_bind 2066..2082 /note=“araI2” −10_signal 2103..2108 /note=“−10” −35_signal 2079..2084 /note=“−35” promoter 2013..2165 /dnas_title=“pBAD” /vntifkey=“30” /label=pBAD CDS 2166..2897 /dnas_title=“dnaQ926” /vntifkey=“4” /label=dnaQ926 conflict 2199..2201 /note=“D12A” conflict 2205..2207 /note=“E14A” RBS 2156..2165 /note=“>sd5 RBS” RBS 2907..2925 /note=“Modified mutS RBS” CDS 2926..3762 /note=“dam (wt)” CDS 3791..4336 /note=“seqA (wt)” RBS 3771..3790 /note=“seqA Native RBS” CDS 4365..4895 /note=“emrR (wt)” RBS 4345..4364 /note=“native emrR RBS” CDS 4924..5178 /note=“PBS2 UGI” RBS 4903..4923 /note=“Native UGI RBS” RBS 5187..5207 /note=“dnaE RBS” CDS 5208..2834 /note=“pmCDA1 (opt)” −10_signal complement (734..739) /note=“−10” −35_signal complement (757..762) /note=“−35” −10_signal (606..611) /note=“−10” −35_signal 582..587 /note=“−35” promoter complement (729..768) /note=“RNA II Promoter (0.93)” promoter 576..615 /note=“RNA I Promoter (0.95)” misc_RNA 618..722 /note=“RNA I” misc_RNA complement (39..725) /note=“RNA II”

(SEQ ID NO: 26) cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc gctgcggaca catacaaagt 60 tacccacaga ttccgtggat aagcagggaa ctaacatgtg aggcaaaaca gcagggccgc 120 gccggtggcg tttttccata ggctccgccc tcctgccaga gttcacataa acagacgctt 180 ttccggtgca tctgtgggag ccgtgaggct caaccatgaa tctgacagta cgggcgaaac 240 ccgacaggac ttaaagatcc ccaccgtttc cggcgggtcg ctccctcttg cgctctcctg 300 ttccgaccct gccgtttacc ggatacctgt tccgcctttc tcccttacgg gaagtgtggc 360 gctttctcat agctcacaca ctggtatctc ggctcggtgt aggtcgttcg ctccaagctg 420 ggctgtaagc aagaactccc cgttcagccc gactgctgcg ccttatccgg taactgttca 480 cttgagtcca acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg 540 gagttcgcag aggatttgtt tagctaaaca cgcggttgct cttgaagtgt gcgccaaagt 600 ccggctacac tggaaggaca gatttggttg ctgtgctctg cgaaagccag ttaccacggt 660 taagcagttc cccaactgac ttaaccttcg atcaaaccac ctccccaggt ggttttttcg 720 tttacagggc aaaagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 780 tctactgaac cgctctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc 340 agcccaggag gaagaggaca tccggtcaaa taaaacgaaa ggctcagtcg aaagactggg 900 cctttcgttt tagacttagg gaccctttat gacaacttga cggctacatc attcactttt 960 tcttcacaac cggcacggaa ctcgctcggg ctggccccgg tgcatttttt aaatacccgc 1020 gagaaataga gttgatcgtc aaaaccaaca ttgcgaccga cggtggcgat aggcatccgg 1080 gaggtgctca aaagcagctt cgcctggctg atacgttggt cctcgcgcca gcttaagacg 1140 ctaatcccta actgctggcg gaaaagatgt gacagacgcg acggcgacaa gcaaacatgc 1200 tgtgcgacgc tggcgatatc aaaattgctg tctgccaggt gatcgctgat gtactgacaa 1260 gcctcgcgta cccgattatc catcggtgga tggagcgact cgttaatcgc ttccatgcgc 1320 cgcagtaaca attgctcaag cagatttatc gccagcagct ccgaatagcg cccttcccct 1380 tgcccggcgt taatgatttg cccaaacagg tcgctgaaat gcggctggtg cgcttcatcc 1440 gggcgaaaga accccgtatt ggcaaatatt gacggccagt taagccattc atgccagtag 1500 gcgcgcggac gaaagtaaac ccactggtga taccattcgc gagcctccgg atgacgaccg 1560 tagtgatgaa tctctcctgg cgggaacagc aaaatatcac ccggtcggca aacaaattct 1620 cgtccctgat ttttcaccac cccctgaccg cgaatggtga gattgagaat ataacctttc 1680 attcccagcg gtcggtcgat aaaaaaatcg agataaccgt tggcctcaat cggcgttaaa 1740 cccgccacca gatgggcatt aaacgagtat cccggcagca ggggatcatt ttgcgcttca 1800 gccatacttt tcatactccc accattcaga gaagaaacca attgtccata ttgcatcaga 1860 cattgccgtc actgcgtctt ttactggctc ttctcgctaa cccaaccggt aaccccgctt 1920 attaaaagca ttctgtaaca aagcgggacc aaagccatga caaaaacgcg taacaaaagt 1980 gtctataatc acggcagaaa agtccacatt gattatttgc acggcgtcac actttgctat 2040 gccatagcat ttttatccat aagattagcg gatcctacct gacgcttttt atcgcaactc 2100 gccatagcat ttttatccat aagattagcg gatcctacct gacgcttttt atcgcaactc 2100 tctactgttt ctccataccc gtttttttgg acgcgtacaa ctcaagtctg acataAAtGA 2160 ccgctatgag cactgcaatt acacgccaga tcgttctcGC TaccGCAacc accggtatga 2220 accagattgg tgcgcactat gaaggccaca agatcattga gattggtgcc gttgaagtgg 2280 tgaaccgtcg cctgacgggc aataacttcc atgtttatct caaacccgat cggctggtgg 2340 atccggaagc ctttggcgta catggtattg ccgatgaatt tttgctcgat aagcccacgt 2400 ttgccgaagt agccgatgag ttcatggact atattcgcgg cgcggagttg gtgatccata 2460 acgcagcgtt cgatatcggc tttatggact acgagttttc gttgcttaag cgcgatattc 2520 cgaagaccaa tactttctgt aaggtcaccg atagccttgc ggtggcgagg aaaatgtttc 2580 ccggtaagcg caacagcctc gatgcgttat gtgctcgcta cgaaatagat aacagtaaac 2640 gaacgctgca cggggcatta ctcgatgccc agatccttgc ggaagtttat ctggcgatga 2700 ccggtggtca aacgtcgatg gcttttgcga tggaaggaga gacacaacag caacaaggtg 2760 aagcaacaat tcagcgcatt gtacgtcagg caagtaagtt acgcgttgtt tttgcgacag 2820 atgaagagat tgcagctcat gaagcccgtc tcgatctggt gcagaagaaa ggcggaagtt 2880 gcctctggcg aacataattt aatatcagta aaccggacat aacccatgaa gaaaaatcgc 2940 gcttttttga agtgggcagg gggcaagtat cccctgcttg atgatattaa acggcatttg 3000 cccaagggcg aatgtctggt tgagcctttt gtaggtgccg ggtcggtgtt tctcaacacc 3060 gacttttctc gttatatcct tgccgatatc aatagcgacc tgatcagtct ctataacatt 3120 gtgaagatgc gtactgatga gtacgtacag gccgcacgcg agctgtttgt tcccgaaaca 3180 aattgcgccg aggtttacta tcagttccgc gaagagttca acaaaagcca ggatccgttc 3240 cgtcgggcgg tactgttttt atatttgaac cgctacggtt acaacggcct gtgtcgttac 3300 aatctgcgcg gtgagtttaa cgtgccgttc ggccgctaca aaaaacccta tttcccggaa 3360 gcagagttgt atcacttcgc tgaaaaagcg cagaatgcct ttttctattg tgagtcttac 3420 gccgatagca tcgcgcgcgc agatgataca tccgtcgtct attgcgatcc gccttataca 3480 ccgctgtctg cgaccgccaa ctttacggcg tatcacacaa acagttttac gcttgaacaa 3340 caagcgcatc tggcggagat cgccgaaggt ctggttgagc gccatattcc agtgctgatc 3600 tccaatcacg atacgatatt aacgcgtgag tggtatcagc gcgcaaaatt gcatgtcgtc 3660 aaagttcgac gcagtataag cagcaacggc ggcacacgta aaaaggtgga cgaactgctg 3720 gctttataca aaccaggagt cgtttcaccc gcaaaaaaat aattcagcta agacactgca 3780 ctggattaag atgaaaacga ttgaagttga tgatgaactc tacagctata ttgccagcca 3840 cactaagcat atcggcgaga gcgcatccaa cattttacgg catatgttga aattttccac 3900 cgcatcacag cctgctgctc cggtgacgaa agaggttcgc gttgcgtcac ctgctatcgt 3960 cgaagcgaag ccggtcaaaa cgattaaaga caaggttcgc gcaatgcgtg aacttctgct 4020 ttcggatgaa tacgcagagc aaaagcgagc ggtcaatcgc tttatgctgc tgttgtctac 4080 actatattct cttgacgccc aggcgtttgc cgaagcaacg gaatcgttgc acggtcgtac 4140 acgcatttac tttgcggcaa atgaacaaac gctgctgaaa aatggtaatc agaccaagcc 4200 gaaacatgtg ccaggcacgc cgtattgggt gatcaccaac accaacaccg gccgtaaatg 4260 cagcatgatc gaacacatca tgcagtcgat gcaattcccg gcggaattga ttgagaaggt 4320 ttgcaaaact atctaacggc tgaaattaat gaggtcatac ccaaatggat agttcgttta 4380 cgcccattga acaaatgcta aaatttcgcg ccagccgcca cgaagatttt ccttatcagg 4440 agatccttct gactcgtctt tgcatgcaca tgcaaagcaa gctgctggag aaccgcaata 4500 aaatgctgaa ggctcagggA attaacgaga cgttgtttat ggcgttgatt acgctggagt 4560 ctcaggaaaa ccacagtatt cagccttctg aattaagttg tgctcttgga tcatcccgta 4620 ccaacacgac gcgtattacc gatgaactgg aaaaacgcgg ttggatcaaa cgtcgtgaaa 4690 gcgataacga tcgccgctgc ctgcatctgc aattaacgga aaaaggtcac gagtttttgc 4740 gcgaggtttt accaccgcag cataactgcc tgcatcaact ctggtccgcg ctcagcacaa 4800 cagaaaaaga tcagctcgag caaatcaccc gcaaattgct ctcccgtctc gaccagatgg 4860 aacaagacgg tgtggttctc gaagcgatga gctaataata caaaaattag gaggaatttc 4920 aacatgacaa atttatctga catcattgaa aaagaaacag gaaaacaact agtgattcaa 4980 gaatcaattc taatgttacc agaagaagta gaggaagtaa ttgggaataa accagaaagt 5040 gatattttag ttcatactgc ttatgatgaa agtacagatg aaaatgtaat gctattaact 5100 tcagatgctc cagaatataa accttgggct ttagtaattc aagacagtaa tggagaaaat 5160 aaaattaaaa tgttataagt cgagattaag taaaccggaa tctgaag   5220           5280           5340           5400           5460           5520           5580           5640           5700           5760           5820   acttaa ttaacggcac tcctcagcca agtcaaaagc ctccgGTcgg 5880 aggcttttga ctacatgccc atggcgttta cgccccgccc tgccactcat cgcagtactg 5940 ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg 6000 aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tagtgaaaac 6060 ggggacgaag aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca 6120 gggattggct gagacgaaaa acatattctc aataaaccct ttagagaaat aggccagatt 6180 ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg 6240 gtattcactc cagagcgata aaaacgtttc agtttactca tggaaaacgg tgtaacaagg 6300 gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgga actccggatg 6360 agcattcatc aggcgagcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt 6420 ctttacggtc tttaaaaagg ccgtaatatc cagctaaacg gtctggttat aggtacattg 6480 agTaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt 6540 ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa 6600 ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac 6660 gtgccaAgcc aaataggccg t 6690

Exemplary Drift Plasmid Sequences

The sequences for exemplary, non-limiting embodiments of drift plasmids, named Drift Plasmids 1-6 (DP 1-6), comprising exemplary drift expression constructs, are provided herein. Table 7 shows the coding DNA sequence (CDS) information for exemplary DP plasmids DP1-6. FIGS. 20-25 provide vector maps of drift plasmids DP1-DP6 (SEQ ID NOs: 27-29, 33-35), respectively. It should be understood that mutagenesis plasmids depicted in FIGS. 26-106, corresponding to SEQ ID NOs: 43-123, can be modified to function as drift plasmids by the inclusion of an anhydrotetracycline (ATc)-dependent drift promoter (e.g., SEQ ID NO: 124) in the construct.

TABLE 7 Coding DNA sequence (CDS) information for exemplary DP plasmids CDS DP1 DP2 DP3 DP4 DP5 DP6 araC 2885-3763 2885-3763 2885-3763 2885-3763 2885-3763 2885-3763 dnaQ926 4124-4855 4124-4855 4124-4855 4124-4855 4124-4855 4124-4855 umuD 4893-5240 — — — — — umuC 5243-6523 — — — — — recA730 6587-7648 — — — — — dam — — 4884-5720 4884-5720 4884-5720 4884-5720 seqA — — — 5749-6294 5749-6294 5749-6294 emrR — — — — — 6323-6853 PBS2 UGI 13 — — — 6323-6577 6882-7136 pmCDA1 13 — — — 6607-7233 7166-7792 DP1 LOCUS  pJC184   6537 bp DNA circular FEATURES    Location/Qualifiers  modified_base 852..866      /note=“USER junction”  terminator  867..911      /note=“rrnB1 transcriptional terminator”  modified_base 919..933      /note=“USER junction”  misc_feature 926..932      /note=“SanDI”      /note=“can be used to exchange f1 origin or selection components”  rep_origin complement(39..777)      /dnas_title=“cloDF13”      /vntifkey=“33”      /label=cloDF13  terminator complement(7673..7708)      /note=“P14/tonB bidirectional terminator”      /note=“termination of cat transcript is slightly weaker      than in opposite direction”  modified_base 7709..7721      /note=“USER junction”  CDS    complement(7722..8381)      /note=“cat (CmR)”      /note=“from pAYCDuet-1”  modified_base 7797..7797      /note=“mutation”      /note=“annotated as a G in pACYCDuet cat marker      annotation, here it is an A, but this mutation is silent      from codon GTC (Val, 25% codon usage) to GTT (Val, 21%      usage), so it should not be of functional relevance”  misc_feature 7666..7672      /note=“Nt-BbvCI”      /note=“nicking endonuclease site that generates overhang”  modified_base 7649..7665      /note=“USER junction”  misc_feature 7651..7658      /note=“PacI”      /note=“can be used to exchange antibiotic resistance marker or selection      components”  promoter  8382..8479      /note=“cat promoter”      /note=“from pACYCDuet-1”  modified_base 8480..8495      /note=“USER junction”  misc_feature 8481..8493      /note=“SfiI”      /note=“can be used to exchange plasmid origin or antibiotic resistance marker”  CDS    complement(2885..3763)      /dnas_title=“araC”      /vntifkey=“4”      /label=araC  misc_feature 2885..2925  promoter  complement(3429..3446)      /dnas_title=“SeqaraC01”      /vntifkey=“30”      /label=SeqaraC01  misc_feature 3861..3861      /dnas_title=“C to A ***”      /vntifkey=“21”      /label=C to A ***  misc_feature 3779..3779      /dnas_title=“A to G ***”      /vntifkey=“21”      /label=A to G ***  promoter  3914..4066      /dnas_title=“pBAD”      /vntifkey=“30”      /label=pBAD  CDS    4124..4855      /dnas_title=“dnaQ926”      /vntifkey=“4”      /label=dnaQ926  promoter  complement(4353..4371)      /dnas_title=“dnaQ02”      /vntifkey=“30”      /label=dnaQ02  promoter  complement(4839..4853)      /dnas_title=“SeqdnaQ03”      /vntifkey=“30”      /label=SeqdnaQ03  CDS    4893..5240      /dnas_title=“umuD”      /vntifkey=“4”      /label=umuD  CDS    5243..6523      /dnas_title=“umuC”      /vntifkey=“4”      /label=umuC  CDS    6587..7648      /dnas_title=“recA730”      /vntifkey=“4”      /label=redA730  misc_feature 7610..7648      /note=“”  misc_feature 2936..22936      /note=“originally an ‘a’ in annotation”  misc_feature 2975..2975      /note=“originally a t in annotation”  misc_feature 3041..3041      /note=“originaily an a in annotation”  misc_feature 3245..3245      /note=“originally a c in annotation”  misc_feature 3410..3410      /note=“originally an a in annotation”  misc_ feature 3569..3569      /note=“originally a g in annotation”  misc_ feature 3716..3716      /note=“originally a g in annotation”  misc_feature 6925..6925      /note=“c in originally annotation”  misc_feature 4051..4051      /note=“”  misc_feature 4037..4017      /note=“”  primer_bind 39..63      /note=“AB711”  primer_bind 164..189      /note=“AB712”  primer_bind 3388_3409      /note=“AB713”  primer_bind 3266..3287      /note=“AB714”  primer_bind 4653..4676      /note=“AB715”  primer_bind 4530..4554      /note=“A9716”  prime_bind 5945..5968      /note=“AB717”  primer_bind 5811_5833      /note=“AB718”  primer_bind 7213..7235      /note=“AB719”  primer_bind 7079..7101      /note=“AB720”  CDS    complement(934..2208)      /dnas_title=“III”      /vntifkey=“4”      /label=III  misc_feature complement(1021..1066)      /note=“modified to remove internal promoter”  primer_bind 1060..1085      /note=“AB691”  primer_bind 1234..1254      /note=“AB424”  primer_bind 1094..1114      /note=“AB423”  misc_feature 2423..2435      /note=“USER linker”  RBS    complement(2209..2222)      /note=“sd8 RBS (from Ringquist and Gold Mol. Micro.      1992)”  modified_base complement (2223..2237)      /note=“USER junction”  misc_feature complement(2229..2236)      /note=“SbfI”  misc_feature complement(2224..2229)      /note=“BsgI”  misc_feature complement(2317..2324)      /note=“”  misc_feature complement(2308..2312)      /note=“”  misc_ feature complement(2297..2297)      /note=“”  primer_bind complement(2305..2345)      /note=“”  primer_bind 2266..2324      /note=“”  protein_bind complement(2278..2296)      /note=“tetR binding site”  terminator complement(2443..2867)      /note=“rrnB1 transcriptional terminator”  primer_bind 2489..2511      /note=“AB428”  primer_bind 2240..2267      /note=“AB721”  misc_feature 2868..2884      /note=“USER linker”  modified_base 789..806      /note=“User Junction”  primer_bind 789..818      /note=“”  primer_bind complement(2847..2884)      /note“”  primer_bind 2868..2912      /note“”  primer_bind complement(777..806)      /note“”  misc_difference 912..918      /note=“cloning scar”  source   1..8495

(SEQ ID NO: 27) 1 cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc gctgcggaca catacaaagt 61 tacccacaga ttccgtggat aagcagggga ctaacatgtg aggcaaaaca gcagggccgc 121 gccggtggcg tttttccata ggctccgccc tcctgccaga gttcacataa acagacgctt 181 ttccggtgca tctgtgggag ccgtgaggct caaccatgaa tctgacagta cgggcgaaac 241 ccgacaggac ttaaagatcc ccaccgtttc cggcgggtcg ctccctcttg cgctctcctg 301 ttccgaccct gccgtttacc ggatacctgt tccgcctttc tcccttacgg gaagtgtggc 361 gctttctcat agctcacaca ctggtatctc ggctcggtgt aggtcgttcg ctccaagctg 421 ggctgtaagc aagaactccc cgttcagccc gactgctgcg ccttatccgg taactgttca 481 cttgaatcca acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg 541 gagttcgcag aggatttgtt tagctaaaca cgcggttgct cttgaagtgt gcgccaaagt 601 ccggctacac tggaaggaca gatttggttg ctgtgctctg cgaaagccag ttaccacggt 661 taagcagttc cccaactgac ttaaccttcg atcaaaccac ctccccaggt ggttttttcg 721 tttacagggc aaaagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 781 tctactgaac cgctctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc 841 agcccaggag gaagaggaca tccggtcaaa taaaacgaaa ggctcagtcg aaagactggg 901 cctttcgttt tGCTGAGGag acttagggac cctttaagac tccttattac gcagtatgtt 961 agcaaacgta gaaaatacat acataaaggt ggcaacatat aaaagaaacg caaagacacc 1021 gcggaacagg ttgatcttat cgcagtcgat actgaactcg taaggtttac cagcgccaaa 1081 gacaaaaggg cgacattcaa ccgattgagg gagggaaggt aaatattgac ggaaattatt 1141 cattaaaggt gaattatcac cgtcaccgac ttgagccatt tgggaattag agccagcaaa 1201 atcaccagta gcaccattac cattagcaag gccggaaacg tcaccaatga aaccatcgat 1261 agcagcaccg taatcagtag cgacagaatc aagtttgcct ttagcgtcag actgtagcgc 1321 gttttcatcg gcattttcgg tcataacccc cttattagcg tttgccatct tttcataatc 1381 aaaatcaccg gaaccagagc caccaccgga accgcctccc tcagagccgc caccctcaga 1441 accgccaccc tcagagccac caccctcaga gccgccacca gaaccaccac cagagccgcc 1501 gccagcattg acaggaggtt gaggcaggtc agacgattgg ccttgatatt cacaaacgaa 1561 tggatcctca ttaaagccag aatggaaagc gcagtctctg aatttaccgt tccagtaagc 1621 gtcatacatg gcttttgatg atacaggagt gtactggtaa taagttttaa cggggtcagt 1681 gccttgagta acagtgcccg tataaacagt taatgccccc tgcctatttc ggaacctatt 1741 attctgaaac atgaaagtat taagaggctg agactcctca agagaaggat taggattagc 1801 ggggttttgc tcagtaccag gcggataagt gccgtcgaga aagttgatat aagtatagcc 1861 cggaataggt gtatcaccgt actcaggagg tttagtaccg ccaccctcag aaccgccacc 1921 ctcagaaccg ccaccctcag agccaccacc ctcattttca gggatagcaa gcccaatagg 1981 aacccatgta ccgtaacact gagtttcgtc accagtacaa actacaacgc ctgtaacatt 2041 ccacagacag ccctcatagt tagcgtaacg atctaaagtt ttgtcgtctt tccagacgtt 2101 agtaaatgaa ttttctgtat ggggttttgc taaacaactt tcaacagttt cdgcggagtg 2161 agaatagaaa agaacaacta aaggaattgc gaataataat tttttcattt tttttttcct 2221 ttactgcacc tgcaggtaat gttgtcctct tgatttctgc gttcaggatt gtcctgctct 1741 attctgaaac atgaaagtat taagaggctg agactcctca agagaaggat taggattagc 1801 ggggttttgc tcagtaccag gcggataagt gccgtcgaga gggttgatat aagtatagcc 1861 cggaataggt gtatcaccgt actcaggagg tttagtaccg ccaccctcag aaccgccacc 1921 ctcagaaccg ccaccctcag agccaccacc ctcattttca gggatagcaa gcccaatagg 1981 aacccatgta ccgtaacact gagtttcgtc accagtacaa actacaacgc ctgtagcatt 2041 ccacagacag ccctcatagt tagcgtaacg atctaaagtt ttgtcgtctt tccagacgtt 2101 agtaaatgaa ttttctgtat ggggttttgc taaacaactt tcaacagttt cagcggagtg 2161 agaatagaaa ggaacaacta aaggaattgc gaataataat tttttcattt tttttttcct 2221 ttactgcacc tgcaggtaat gttgtcctct tgatttctgc gttcaggatt gtcctgctct 2281 ctatcactga tagggatgaa ctgttaatac aatttgcgtg ccaatttttt atctttttga 2341 tttataaaga tctgattgaa gaatcaacag caacatgcca ggatgagtta gcgaattaca 2401 ctaacaagtg gcgaatttca tcacggagcc aatgtcctca gcgagtttgt agaaacgcaa 2461 aaaggccatc cgtcaggatg gccttctgct taatttgatg cctggcagtt catggcgggc 2521 gtcctgcccg ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cccggcggat 2581 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt 2641 tcgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagac 2701 cccacactac catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg 2761 accaccgcgc tactgccgcc aggcaaattc tgttttatca gaccgcttct gcgttctgat 2821 ttaatctgta tcaggctgaa aatcttctct catccgccaa aacagccagg gccctactga 2881 ctgtttatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact 2941 cgctcgggct ggccccggtg catttttcaa atacccgcga gaaatagagt tgatcgtcaa 3001 aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg 3061 cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga 3121 aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa 3181 aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca 3241 tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca 3301 gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc 3361 caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg 3421 caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc 3481 actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg 3541 ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc 3601 cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa 3661 aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa 3721 acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccac 3781 cattcagaga agaaaccaat tgtccatact gcatcagaca ttgccgtcac tgcgtctttt 3841 actggctctt ctcgctaacc caaccggtaa ccccgcttat taaaagcatt ctgtaacaaa 3901 gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag 3961 tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa 4021 gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct ccatacccgt 4081 ttttttggac gcgtacaact caagtctgac ataaatgacc gctatgagca ctgcaattac 4141 acgccagatc gttctcgcta ccgcaaccac cggtatgaac cagattggtg cgcactatga 4201 aggccacaag atcattgaga ttggtgccgt tgaagtggtg aaccgtcgcc tgacgggcaa 4261 taacttccat gtttatctca aacccgatcg gctggtggat ccggaagcct ttggcgtaca 4321 tggtattgcc gatgaatttt tgctcgataa gcccacgttt gccgaagtag ccgatgagtt 4381 catggactat attcgcggcg cggagttggt gatccataac gcagcgttcg atatcggctt 4441 tatggactac gagttttcgt tgcttaagcg cgatattccg aagaccaata ctttctgtaa 4501 ggtcaccgat agccttgcgg tggcgaggaa aatgtttccc ggtaagcgca acagcctcga 4561 tgcgttatgt gctcgctacg aaatagataa cagtaaacga acgctgcacg gggcattact 4621 cgatgcccag atccttgcgg aagtttatct ggcgatgacc ggtggtcaaa cgtcgatggc 4681 ttttgcgatg gaaggagaga cacaacagca acaaggtgaa gcaacaattc agcgcattgt 4741 acgtcaggca agtaagttac gcgttgtttt tgcgacagat gaagagattg cagctcatga 4801 agcccgtctc gatctggtgc agaagaaagg cggaagttgc ctctggcgag cataaaccgg 4861 tatgcctcac acaggaaaca gaattcatta ccatgggctt tccttcaccg gcagcagatt 4921 acgttgaaca gcgcatcgat ctgaatcaac tgttgatcca gcatcccagc gcgacttact 4981 tcgtcaaagc aagtggtgat tctatgattg atggtggaat tagtgacggt gatttactga 5041 ttgtcgatag cgctattacc gccagccatg gtgatattgt catcgctgct gttgacggcg 5101 agtttacggt gaaaaaattg caactacgcc cgacggtaca gcttattccc atgaacagcg 5161 cgtactcgcc cattaccatc agtagtgaag atacgctgga tgtctttggt gtggtgatcc 5221 acgtcgttaa ggcgatgcgc tgatgtttgc cctctgtgat gtaaacgcgt tttatgccag 5281 ctgtgagacg gtgtttcgcc ctgatttatg gggtaaaccg gtggttgtgc tatcgaataa 3341 tgacggttgc gttatcgccc gaaacgctga ggcaaaggcg cttggcgtta aaatgggcga 5401 tccctggttc aaacaaaaag atctgtttcg tcgctgtggc gtggtttgct ttagcagcaa 5461 ttatgagctt tacgcagaca tgagcaatcg ggtgatgtcg acgctggaag agctatcgcc 5521 ccgcgtcgag atttacagta ccggtatgcc tattgatgag gcattctgcg atctgacagg 5581 tatgcgtaat tgtcgcgatc tgactgattt tggcagagaa attcgcgcaa cggtgctaca 5641 acgtacccat cttactgttg gtgtggggat cgcccagacc aaaacgctgg ctaagcttgc 5701 caatcatgcg gcaaaaaaat ggcagcggca gacgggtggg gtggtggatt tatcaaatct 5761 ggaacgccag cgtaaattaa tgtctgccct ccccgtggat gacgtctggg ggattggacg 5821 gcggatcagc aaaaaactgg acgcgatggg gatcaaaacc gttctcgatt tggcggatac 5881 agatatccgg tttatccgta aacattttaa tgtcgtgctc gaaagaacgg tgcgtgaact 5941 gcgcggcgaa ccctgtttgc aactggaaga gtttgcaccg acgaagcagg aaattatctg 6001 ttcccgctcg tttggtgaac gcatcacgga ttatccgtcg atgcggcagg ccatttgtag 6061 ttacgctgcc cgggcggcgg aaaaacttcg cagcgagcat caatattgtc gattaatttc 6121 cacgtttatt aagacgtcac catttgcgct caatgaacct tattacggca atagcgcgtc 6181 ggtaaaactg ctgacgccca ctcaggacag cagggatatc attaacgctg ctacgcgatc 6241 tctggatgcc atctggcaag cgggccatcg ttaccaaaaa gcgggcgtga tgctggggga 6301 tttcttcagt cagggagtcg cgcagctcaa tttattcgat gacaacgcac cgcgccccgg 6361 gagtgagcaa ttgatgacgg taatggatac actgaatgct aaagagggca gaggaacact 6421 ctattttgcc gggcagggga tccagcaaca atggcagatg aagcgagcca tgctttcacc 6481 acgttataca acgcgaagtt ctgatttact gagggtcaaa taaatatagc ggcaggaaaa 6541 aagcgatccc gcatatccgg tattacccgg catgacagga gtaaaaatgg ctatcgacga 6601 aaacaaacag aaagcgttgg cggcagcact gggccagatt gagaaacaat ttggtaaagg 6661 ctccatcatg cgcctgggtg aagaccgttc catggatgtg aaaaccatct ctaccggttc 6721 gctttcactg gatatcgcgc ttggggcagg tggtctgccg atgggccgta tcgtcgaaat 6781 ctacggaccg gaatcttccg gtaaaaccac gctgacgctg caggtgatcg ccgcagcgca 6841 gcgtgaaggt aaaacctgtg cgtttatcga tgctgaacac gcgctggacc caatctacgc 6901 acgtaaactg ggcgtcgata tcgataacct gctgtgctcc cagccggaca ccggcgagca 6961 ggcactggaa atctgtgacg ccctggcgcg ttctggcgca gtagacgtta tcgtcgttga 7021 ctccgtggcg gcactgacgc cgaaagcgga aatcgaaggc gaaatcggcg actctcacat 7081 gggccttgcg gcacgtatga tgagccaggc gatgcgtaag ctggcgggta acctgaagca 7141 gtccaacacg ctgctgatct tcatcaacca gatccgtatg aaaattggtg tgatgttcgg 7201 taacccggaa accactaccg gtggtaacgc gctgaaattc tacgcctctg ttcgtctcga 7261 catccgtcgt atcggcgcgg tgaaagaggg cgaaaacgtg gtgggtagcg aaacccgcgt 7321 gaaagtggtg aagaacaaaa tcgctgcgcc gtttaaacag gctgaattcc agatcctcta 7381 cggcgaaggt atcaacttct acggcgaact ggttgacctg ggcgtaaaag agaagctgat 7441 cgagaaagca ggcgcgtggt acagctacaa aggtgagaag atcggtcagg gtaaagcgaa 7501 tgcgactgcc tggctgaaag ataacccgga aaccgcgaaa gagatcgaga agaaagtacg 7561 tgagttgctg ctgagcaacc cgaactcaac gccggatttc tctgtagatg atagcgaagg 7621 cgtagcagaa actaacgaag atttttaaac ttaattaacg gcactcctca gccaagtcaa 7681 aagcctccga ccggaggctt ttgactacat gcccatggcg tttacgcccc gccctgccac 7741 tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc atcacaaacg 7801 gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg 7861 cccatagtga aaacgggggc gaagaagtcg tccatattgg ccacgtttaa atcaaaaccg 7921 gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa cccLttaggg 7981 aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc 8041 cggaaatcgt cgtagtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa 8101 acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata 8161 cggaactccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac 8221 ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg 8281 ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg 8341 gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct 8401 gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt atggtgaaag 8461 ttggaacctc ttacgtgcca Agccaaatag gccgt (SEQ ID NO: 28) 1 cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc gctgcggaca catacaaagt 61 tacccacaga ttccgtggat aagcagggga ctaacatgtg aggcaaaaca gcagggccgc 121 gccggtggcg tttttccata ggctccgccc tcctgccaga gttcacataa acagacgctt 181 ttccggtgca tctgtgggag ccgtgaggct caaccatgaa tctgacagta cgggcgaaac 241 ccgacaggac ttaaagatcc ccaccgtttc cggcgggtcg ctccctcttg cgctctcctg 301 ttccgaccct gccgtttacc ggatacctgt tccgcctttc tcccttacgg gaagtgtggc 361 gcttcctcat agctcacaca ctggtatctc ggctcggtgt aggtcgttcg ctccaagctg 421 ggctgtaagc aagaactccc cgttcagccc gactgctgcg ccttatccgg taactgttca 481 cttgagtcca acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg 541 gagttcgcag aggatttgtt tagctaaaca cgcggttgct cttgaagtgt gcgccaaagt 601 ccggctacac tggaaggaca gatttggttg ctgtgctctg cgaaagccag ttaccacggt 661 taagcagttc cccaactgac ttaaccttcg atcaaaccac ctccccaggt ggttttttcg 721 tttacagggc aaaagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 781 tctactgaac cgctctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc 841 agcccaggag gaagaggaca tccggtcaaa taaaacgaaa ggctcagtcg aaagactggg 901 cctttcgttt tGCTGAGGag acttagggac cctttaagac tccttattac gcagtatgtt 961 agcaaacgta gaaaatacat acataaaggt ggcaacatat aaaagaaacg caaagacacc 1021 gcggaacagg ttgatcttat cgcagtcgat actgaactcg taaggtttac cagcgccaaa 1081 gacaaaaggg cgacattcaa ccgattgagg gagggaaggt aaatattgac ggaaattatt 1141 cattaaaggt gaattatcac cgtcaccgac ttgagccatt tgggaattag agccagcaaa 1201 atcaccagta gcaccattac cattagcaag gccggaaacg tcaccaatga aaccatcgat 1261 agcagcaccg taatcagtag cgacagaatc aagtttgcct ttagcgtcag actgtagcgc 1321 gttttcatcg gcattttcgg tcatagcccc cttattagcg tttgccatct tttcataatc 1381 aaaatcaccg gaaccagagc caccaccgga accgcctccc tcagagccgc caccctcaga 1441 accgccaccc tcagagccac caccctcaga gccgccacca gaaccaccac cagagccgcc 1501 gccagcattg acaggaggtt gaggcaggtc agacgattgg ccttgatatt cacaaacgaa 1561 tggatcctca ttaaagccag aatggaaagc gcagtctctg aatttaccgt tccagtaagc 1621 gtcatacatg gcttttgatg atacaggagt gtactggtaa taagttttaa cggggtcagt 1681 gccttgagta acagtgcccg tataaacagt taatgccccc tgcctatttc ggaacctatt 1741 attctgaaac atgaaagtat taagaggctg agactcctca agagaaggat taggattagc 1801 ggggttttgc tcagtaccag gcggataagt gccgtcgaga gggttgatat aagtatagcc 1861 cggaataggt gtatcaccgt actcaggagg tttagtaccg ccaccctcag aaccgccacc 1921 ctcagaaccg ccaccctcag agccaccacc ctcattttca gggatagcaa gcccaatagg 1981 aacccatgta ccgtaacact gagtttcgtc accagtacaa actacaacgc ctgtagcatt 2041 ccacagacag ccctcatagt tagcgtaacg atctaaagtt ttgtcgtctt tccagacgct 2101 agtaaatgaa ttttctgtat ggggttttgc taaacaactt tcaacagttt cagcggagtg 2161 agaatagaaa ggaacaacta aaggaattgc gaataataat tttttcattt tttttttcct 2221 ttactgcacc tgcaggtaat gttgtcctct tgatttctgc gttcaggatt gtcctgctct 2281 ctatcactga tagggatgaa ctgttaatac aatttgcgtg ccaatttttt atctttttga 2341 tttataaaga tctgattgaa gaatcaacag caacatgcca ggatgagtta gcgaattaca 2401 ctaacaagtg gcgaatttca tcacggagcc aatgtcctca gcgagtttgt agaaacgcaa 2461 aaaggccatc cgtcaggatg gccttctgct taatttgatg cctggcagtt tatggcgggc 2521 gtcctgcccg ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cccggcggat 2581 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt 2641 tcgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagac 2701 cccacactac catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg 2761 accaccgcgc tactgccgcc aggcaaattc tgttttatca gaccgcttct gcgttctgat 2821 ttaatctgta tcaggctgaa aatcttctct catccgccaa aacagccagg gccctactga 2881 ctgtttatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact 2941 cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa 3001 aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg 3061 cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga 3121 aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa 3181 aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca 3241 tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca 3301 gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc 3361 caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg 3421 caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc 3481 actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg 3541 ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc 3601 cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa 3661 aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa 3721 acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccac 3781 cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt 3841 actggctctt ctcgctaacc caaccggtaa ccccgcttat taaaagcatt ctgtaacaaa 3901 gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag 3961 tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa 4021 gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct ccatacccgt 4081 ttttttggac gcgtacaact caagtctgac ataaatgacc gctatgagca ctgcaattac 4141 acgccagatc gttctcgcta ccgcaaccac cggtatgaac cagattggtg cgcactatga 4201 aggccacaag atcattgaga ttggtgccgt tgaagtggtg aaccgtcgcc tgacgggcaa 4261 taacttccat gtttatctca aacccgatcg gctggtggat ccggaagcct ttggcgtaca 4321 tggtattgcc gatgaatttt tgctcgataa gcccacgttt gccgaagtag ccgatgagtt 4381 catggactat attcgcggcg cggagttggt gatccataac gcagcgttcg atatcggctt 4441 tatggactac gagttttcgt tgcttaagcg cgatattccg aagaccaata ctttctgtaa 4501 ggtcaccgat agccttgcgg tggcgaggaa aatgtttccc ggtaagcgca acagcctcga 4561 tgcgttatgt gctcgctacg aaatagataa cagtaaacga acgctgcacg gggcattact 4621 cgatgcccag atccttgcgg aagtttatct ggcgatgacc ggtggtcaaa cgtcgatggc 4681 ttttgcgatg gaaggagaga cacaacagca acaaggtgaa gcaacaattc agcgcattgt 4741 acgtcaggca agtaagttac gcgttgtttt tgcgacagat gaagagattg cagctcatga 4801 agcccgtctc gatctggtgc agaagaaagg cggaagttgc ctctggcgag cataaactta 4861 attaacggca ctcctcagcc aagtcaaaag cctccgaccg gaggcttttg actacatgcc 4921 catggcgttt acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt 4981 ctgccgacat agaagccatc acaaacagca tgatgaacct aaatcgccag cggcatcagc 5041 accttatcgc cttgcgtata atatttggcc atagtgaaaa cgagggcgaa gaagttgtcc 5101 atattggcca cgtttaaatc aaaactgatg aaactcaccc agggattggc tgagacgaaa 5161 aacatattct caataaaccc tttagaaaaa taggccaggt tttcaccgta acacgccaca 5221 tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat 5281 gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc 5341 accagctcac cgtctttcat tgccatacga aactccagat aagcaticat caggcaagca 5401 agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag 5461 gccgtaatat ccagctgaac ggtctgatta taggtacatt gagcaactga ctgaaatgcc 5521 tcaaaatatt ctttacgatg ccattaagat atatcaacgg tagtatgatc agtgattttt 5581 ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacgcccggt 5641 agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccaAgc caaataggcc 5701 gt (SEQ ID NO: 29) 1 cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc gctgcggaca catacaaagt 61 tacccacaga ttccgtggat aagcaaagga ctaacatgtg aagcaaaaca gcaggaccgc 121 gccggtgacg tttttccata ggctccgccc tcctgccaga gttcacataa acagacgctt 181 ttccggtgca tctgtaggag ccgtgaggct caaccatgaa tctgacagta cgggcgaaac 241 ccgacaggac ttaaagatcc ccaccctttc cggcggatcg ctccctcttg cgctctcctg 301 ttccgaccct gccgtttacc ggatacctgt tccgcctttc tcccttacgg gaagtgtggc 361 gctttctcat agctcacaca ctggtatctc ggctcggtgt aggtcgttcg ctccaagctg 421 ggctgtaagc aagaactccc cattcaaccc gactgctgcg ccttatccgg taactcttca 481 cttaaatcca acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg 541 gagttcgcag aggatttgtt tagctaaaca cgcggttgct cttgaagtgt gcgccaaagt 601 ccggctacac tggaaggaca gatttgcttg ctgtgctctg caaaagccag ttaccacggt 661 taagcagttc cccaactgac ttaaccttcg atcaaaccac ctccccaggt gattttttcg 721 tttacagggc aaaagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 781 tctactgaac cgctctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc 841 agcccaggag gaagaggaca tccgatcaaa taaaacgaaa ggctcagtcg aaagactggg 901 cctttcgtgg tGCTGAGGag acttagggac cctttaagac tccttattac gcagtatgtt 961 agcaaacgta gaaaatacat acataaaggt ggcaacatat aaaagaaacg caaagacacc 1021 gcggaacagg ttgatcttat cgcagtcgat actgaactcg taaggtttac cagcgccaaa 1081 gacaaaaggg cgacattcaa ccgattgagg gaaagaaggt aaatattgac ggaaattatt 1141 cattaaaggt gaattatcac cgtcaccgac ttgagccatt taggaattag agccagcaaa 1201 atcaccagta gcaccattac cattagcaag gccggaaacg tcaccaatga aaccatcgat 1261 agcagcaccg taatcagtaa cgacagaatc aactttgcct ttagcgtcaa actgtagcgc 1321 gttttcatcg gcattttcgg tcatagcccc cttattagcg tttgccatct tttcataatc 1381 aaaatcaccg gaaccagagc caccaccgga accgcctccc tcagagccgc caccctcaga 1441 accaccaccc tcagagccac caccctcaga gccaccacca gaaccaccac cagagccgcc 1501 gccagcattg acaggaggtt gaggcaggtc agacgattgg ccttgatatt cacaaacgaa 1561 tggatcctca ttaaagccag aatggaaagc gcagtctctg aatttaccgt tccagtaagc 1621 gtcatacatg gcttttgata atacaggagt gtactggtaa taagttttaa cgaggtcagt 1681 gccttgagta acaatgcccg tataaacagt taatgccccc tgcctatttc ggaacctatt 1741 attctgaaac atgaaagtat taagaggctg agactcctca agagaaggat taggattagc 1801 ggggttttgc tcagtaccag gcggataagt gccgtcgaga gggttgatat aagtatagcc 1861 cggaataggt gtatcaccgt actcaggagg tttagtaccg ccaccctcag aaccgccacc 1921 ctcagaaccg ccaccctcag agccaccacc ctcattttca gggatagcaa gcccaatagg 1981 aacccatgta ccgtaacact gagtttcgtc accagtacaa actacaacgc ctgtagcatt 2041 ccacagacag ccctcatagt tagcgtaacg atctaaagtt ttgtcgtctt tccagacgtt 2101 agtaaatgaa ttttctgtat gaggttttgc taaacaactt tcaacagttt cagcgaagtg 2161 agaatagaaa ggaacaacta aaggaattgc gaataataat tttttcattt tttttttcct 2221 ttactgcacc tgcaggtaat gttgtcctct tgatttctgc gttcaggatt gtcctgctct 2281 ctatcactga tagggatgaa ctgttaatac aatttgcgtg ccaatttttt atctttttga 2341 tttataaaga tctgattgaa gaatcaacag caacatgcca ggatgagtta gcgaattaca 2401 ctaacaagtg gcgaatttca tcacggagcc aatgtcctca gcgagtttgt agaaacgcaa 2461 aaaggccatc cgtcaggatg gccttctgct taatttgatg cctggcagtt tataacaaac 2521 gtcctgcccg ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cttggcggat 2581 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt 2641 tcgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagac 2701 cccacactac catcgacgct acggcgtttc acttctgagt tcggcatggg gtcagatgga 2761 accaccgcgc tactgccgcc aggcaaattc tgttttatca gaccgcttct gcgttctgat 2821 ttaatctgta tcaggctgaa aatcttctct catccgccaa aacagccagg gccctactga 2881 ctgtttatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacgaaact 2941 cgctcgggct ggccccaatg cattttttaa atacccgcga gaaatagagt tgatcgtcaa 3001 aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg 3061 cctggctgat acgttcggtc tcgcgccagc ttaagacgct aatccctaac tgctgacgga 3121 aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgcta acgatatcaa 3181 aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca 3241 tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca 3301 gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc 3361 caaacaggtc gctgaaatgc gactggtgcg cttcatccgg gcgaaagaac cccgtattgg 3421 caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc 3481 actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg 3541 ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc 3601 cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa 3661 aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa 3721 acgagtatcc cggcagcaaa ggatcatttt gcacttcagc catacttttc atactcccac 3781 cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcatctttt 3841 actggctctt ctcgctaacc caaccggtaa ccccgcttat taaaagcatt ctgtaacaaa 3901 gcaagaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag 3961 tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa 4021 gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct ccatacccgt 4081 ttttttggac gcgtacaact caagtctgac ataaatgacc gctatgagca ctgcaattac 4141 acgccagatc gttctcGCTa ccGCAaccac cggtatgaac cagattgatg cgcactatga 4201 aggccacaag atcattgaga ttgatgccgt tgaagtggtg aaccgtcgcc tgacgggcaa 4261 taacttccat gtttatctca aacccgatcg gctggtggat ccggaagcct ttggcgtaca 4321 tggtattgcc gatgaatttt tgctcgataa gcccacgttt gccgaagtag ccgatgagtt 4381 catggactat attcgcggcg cggagttggt gatccataac gcagcgttcg atatcggctt 4441 tatggactac gagttttcgt tgcttaagcg cgatattccg aagaccaata ctttctgtaa 4501 ggtcaccgat agccttgcgg tggcaaggaa aatgtttccc ggtaagcgca acagcctcga 4561 tgcgttatgt actcgctacg aaataaataa cagtaaacga acgctgcacg gggcattact 4621 cgatgcccag atccttgcgg aagtttatct ggcgatgacc ggtggtcaaa cgtcgatggc 4681 ttttgcgatg gaaggagaga cacaacagca acaaggtgaa gcaacaattc agcgcattgt 4741 acgtcaggca agtaagttac gcgttgtttt tgcgacagat aaagagattg cagctcatga 4801 agcccgtctc gatctggtgc agaagaaagg cggaagttgc ctctggcgag cataatttaa 4861 tatcagtaaa ccggacataa cccatgaaga aaaatcgcgc ttttttgaag tgggcagggg 4921 gcaagtatcc cctgcttgat gatattaaac ggcatttgcc caagggcgaa tgtctggttg 4981 agccttttgt aggtgccggg tcggtgtttc tcaacaccga cttttctcgt tatatccttg 5041 ccgatatcaa tagcgacctg atcagtctct ataacattgt gaagatgcgt actgatgagt 5101 acgtacaggc cgcacgcgag ctgtttgttc ccgaaacaaa ttgcgccgag gtttactatc 5161 agttccgcga agagttcaac aaaagccagg atccgttccg tcgggcggta ctgtttttat 5221 atttgaaccg ctacggttac aacggcctgt gtcgttacaa tctgcgcggt gagtttaacg 5281 tgccgttcgg ccgctacaaa aaaccctatt tcccggaagc agagttgtat cacttcgctg 5341 aaaaagcgca gaatgccttt ttctattgtg agtcttacgc cgatagcalg gcgcgcgcag 5401 atgatgcatc cgtcgtctat tgcgatccgc cttatgcacc gctgtctgcg accgccaact 5461 ttacggcgta tcacacaaac agttttacgc ttgaacaaca agcgcatctg gcggagatcg 5521 ccgaaggtct ggttgagcgc catattccag tgctgatctc caatcacgat acgatgttaa 5581 cgcgtgagtg gtatcagcgc gcaaaattgc atgtcgtcaa agttcgacgc agtataagca 5641 gcaacggcgg cacacgtaaa aaggtggacg aactgctggc tttgtacaaa ccaggagtcg 5701 tttcacccgc gaaaaaataa acttaattaa cggcactcct cagccaagtc aaaagcctcc 5761 gaccggaggc ttttgactac atgcccatgg cgtttacgcc ccgccctgcc actcatcgca 5821 gtaccgttgt aattcattaa gcattctgcc gacatggaag ccatcacaaa cggcatgatg 5881 aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt tgcccatagt 5941 gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac tggtgaaact 6001 cacccaggga ttggctgaga cgaaaaacat attctcaata aaccctttag ggaaataggc 6061 caggttttca ccgtaacacg ccacatcttg cgaatatatg tgtagaaact gccggaaatc 6121 gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta 6181 acaagggtga acactatccc atatcaccag ctcaccgtct ttcattgcca tacggaactc 6241 cggatgagca ttcatcaggc gggcaagaat gtgaataaag gccggataaa acttgtgctt 6301 atttttcttt acggtcttta aaaaggccgt aatatccagc tgaacggtct ggttataggt 6361 acattgagca actgactgaa atgcctcaaa atgttcttta cgatgccatt gggatatatc 6421 aacggtggta tatccagtga tttttttctc cattttagct tccttagctc ctgaaaatct 6481 cgataactca aaaaatacgc ccggtagtga tcttatttca ttatggtgaa agttggaacc 6541 tcttacgtgc caAgccaaat aggccgt (SEQ ID NO: 33) 1 cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc gctgcggaca catacaaagt 61 tacccacaga ttccgtggat aagcagggga ctaacatgtg aggcaaaaca gcagggccgc 121 gccggtggcg tttttccata ggctccgccc tcctgccaga gttcacataa acagacgctt 181 ttccggtgca tctgtgggag ccgtgaggct caaccatgaa tctgacagta cgggcgaaac 241 ccgacaggac ttaaagatcc ccaccgtttc cggcgggtcg ctccctcttg cgctctcctg 301 ttccgaccct gccgtttacc ggatacctgt tccgcctttc tcccttacgg gaagtgtggc 361 gctttctcat agctcacaca ctggtatctc ggctcggtgt aggtcgttcg ctccaagctg 421 ggctgtaagc aagaactccc cgttcagccc gactgctgcg ccttatccgg taactgttca 481 cttgagtcca acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg 541 gagtccgcag aggatttgtt tagctaaaca cgcggttgct cttgaagtgt gcgccaaagt 601 ccggctacac tggaaggaca gatttggttg ctgtgctctg cgaaagccag ttaccacggt 661 taagcagttc cccaactgac ttaaccttcg atcaaaccac ctccccaggt ggttttttcg 721 tttacagggc aaaagattac gcgcagaaaa aaaggacctc aagaagatcc cttgatcttt 781 tctactgaac cgctctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc 841 agcccaggag gaagaggaca tccggtcaaa taaaacgaaa ggctcagtcg aaagactggg 901 cctttcgttt tGCTGAGGag acttagggac cctttaagac tccttattac gcagtatgtt 961 agcaaacgta gaaaatacat acataaaggt ggcaacatat aaaagaaacg caaagacacc 1021 gcggaacagg ttgatcttat cgcagtcgat actgaactcg taaggtttac cagcgccaaa 1081 gacaaaaggg cgacattcaa ccgattgagg gagggaaggt aaatattgac ggaaattatt 1141 cattaaaggt gaattatcac cgtcaccgac ttgagccatt tgggaattag agccagcaaa 1201 atcaccagta gcaccattac cattagcaag gccggaaacg tcaccaatga aaccatcgat 1261 agcagcaccg taatcagtag cgacagaatc aagtttgcct ttagcgtcag actgtagcgc 1321 gttttcatcg gcattttcgg tcatagcccc cttattagcg tttgccatct tttcataatc 1381 aaaatcaccg aaaccagagc caccaccgga accgcctccc tcagagccgc caccctcaga 1441 accgccaccc tcagagccac caccctcaga gccgccacca gaaccaccac cagagccgcc 1501 gccagcattg acaggaggtt gaggcaggtc agacgattgg ccttgatatt cacaaacgaa 1561 tggatcctca ttaaagccag aatggaaagc gcagtctctg aatttaccgt tccagtaagc 1621 gtcatacatg gcttttgatg atacaggagt gtactggtaa taagttttaa cggggtcagt 1681 gccttgagta acagtgcccg tataaacagt taatgccccc tgcctatttc ggaacctatt 1741 attctgaaac atgaaagtat taagaggctg agactcctca aaagaaggat taggattagc 1801 gggattttgc tcagtaccag acggataagt gccgtcgaga gggttgatat aagtatagcc 1861 Aggaataggt gtatcaccgt actcaggagg tttagtaccg ccaccctcag aaccgccacc 1921 ctcagaaccg ccaccctcag agccaccacc ctcattttca aagatagcaa gcccaatagg 1981 aacccatgta ccgtaacact gagtttcgtc accagtacaa actacaacgc ctgtagcatt 2041 ccacagacag ccctcatagt tagcgtaacg atctaaagtt ttgtcgtctt tccagacgtt 2101 agtaaatgaa ttttctgtat gggattttgc taaacaactt tcaacagttt cagcggagtg 2161 agaatagaaa ggaacaacta aaggaattgc gaataataat tttttcattt tttttttcct 2221 ttactgcacc tgcaggtaat gttgtcctct tgatttctgc gttcaggatt gtcctgctct 2281 ctatcactga tagggatgaa ctgttaatac aatttgcgtg ccaatttttt atctttttga 2341 tttataaaga tctgattgaa gaatcaacag caacatgcca ggatgagtta gcgaattaca 2401 ctaacaagtg gcgaatttca tcacggagcc aatatcctac gcgagtttat agaaacgcaa 2461 aaaggccatc cgtcaggatg gccttctgct taatttgatg cctggcagtt tatggcgggc 2521 gtcctgcccg ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cccggcggat 2581 ttatcctact caggagagca ttcaccgaca aacaacagat aaaatgaaaa gcccagtctt 2641 tcgactgagc cccccgtttt atttgatgcc tggcagttcc ctactctcgc atggggagac 2701 cccacactac catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg 2761 accaccgcgc tactgccgcc aggcaaattc tattttatca gaccgcttct gcgttctgat 2821 ttaatctgta tcaggctgaa aatcttctct catccgccaa aacagccagg gccctactga 2881 ctgtttatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact 2941 cgctcgagct ggccccggtc cattttttaa atacccgcga gaaatagagt tgatcgtcaa 3001 aaccaacatt gcgaccgacg gtggcgatag gcatccgggt gatactcaaa agcagcttcg 3061 cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga 3121 aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacactg gcgatatcaa 3181 aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca 3241 tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca 3301 gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcatta atgatttgcc 3301 caaacaggtc gctgaaatgc ggctgatacg cttcatccgg gcaaaagaac cccgtattgg 3421 caaatattga cggccagtta agccattcat gccagtaggc acgcggacga aagtaaaccc 3481 actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg 3541 ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc 3601 cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcaataa 3661 aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa 3721 acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccac 3781 cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt 3841 actggctctt ctcgctaacc caaccggtaa ccccgcttat taaaagcatt ctgtaacaaa 3901 gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag 3961 tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa 4021 gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct ccatacccgt 4081 ttttttggac gcgtacaact caagtctgac ataaatgacc gctatgagca ctgcaattac 4141 acgccagatc gttctcGCTa ccGCAaccac cggtatgaac cagattggtg cgcactatga 4201 aggccacaag atcattgaga ttggtgccgt tgaagtggtg aaccgtcgcc tgacgggcaa 4261 taacttccat gtttatctca aacccgatcg gctggtggat ccggaagcct ttggcgtaca 4321 tggtattgcc gatgaatttt tgctcgataa gcccacgttt gccgaagtag ccgatgagtt 4381 catggactat attcgcggcg cggagttggt gatccataac gcagcgttcg atatcggctt 4441 tatggactac gagttttcgt tgcttaagcg cgatattccg aagaccaata ctttctgtaa 4501 ggtcaccgat agccttgcgg tggcgaggaa aatgtttccc ggtaagcgca acagcctcga 4561 tgcgttatgt gctcgctacg aaatagataa cagtaaacga acgctgcacg gggcattact 4621 cgatgcccag atccttgcgg aagtttatct ggcgatgacc ggtggtcaaa cgtcgatggc 4681 ttttgcgatg gaaggagaga cacaacagca acaaggtgaa gcaacaattc agcgcattgt 4741 acgtcaggca agtaagttac gcgttgtttt tgcgacagat gaagagattg cagctcatga 4801 agcccgtctc gatctggtgc agaagaaagg cggaagttgc ctctggcgag cataatttaa 4861 tatcagtaaa ccggacataa cccatgaaga aaaatcgcgc ttttttgaag tgggcagggg 4921 gcaagtatcc cctgcttgat gatattaaac ggcatttgcc caagggcgaa tgtctggttg 4981 agccttttgt aggtgccggg tcggtgtttc tcaacaccga ctcttctcgt tatatccttg 5041 ccgatatcaa tagcgacctg atcagtctct ataacattgt gaagatgcgt actgatgagt 5101 acgtacaggc cgcacgcgag ctgtttgttc ccgaaacaaa ttgcgccgag gtttactatc 5161 agttccgcga agagttcaac aaaagccagg atccgttccg tcgggcggta ctgtttttat 5221 atttgaaccg ctacggttac aacggcctgt gtcgttacaa tctgcgcggt gagtttaacg 5281 tgccgttcgg ccgctacaaa aaaccctatt tcccggaagc agagttgtat cacttcgctg 5341 aaaaagcgca gaacgccttt ttctattgtg agtcttacgc cgatagcatg gcgcgcgcag 5401 atgatgcatc cgtcgtctat tgcgatccgc cttatgcacc gctgtctgcg accgccaact 5461 ttacggcgta tcacacaaac agttttacgc ttgaacaaca agcgcatctg gcggagatcg 5521 ccgaaggtct ggttgagcgc catattccag tgctgatctc caatcacgat acgatgttaa 5581 cgcgtgagtg gtatcagcgc gcaaaattgc atgtcgtcaa agttcgacgc agtataagca 5641 gcaacggcgg cacacgtaaa aaggtggacg aactgctggc tttgtacaaa ccaggagtcg 5701 tttcacccgc gaaaaaataa ttcagctaag acactgcact ggattaagat gaaaacgatt 5761 gaagttgatg atgaactcta cagctatatt gccagccaca ctaagcatat cggcgagagc 5821 gcatccgaca ttttacggcg tatgttgaaa ttttccgccg catcacagcc tgctgctccg 5881 gtgacgaaag aggttcgcgt tgcgtcacct gctatcgtcg aagcgaagcc ggtcaaaacg 5941 attaaagaca aggttcgcgc aatgcgtgaa cttctgcttt cggatgaata cgcagagcaa 6001 aagcgagcgg tcaatcgctt tatgctgctg ttgtctacac tatattctct tgacgcccag 6061 gcgtttgccg aagcaacgga atcgttgcac ggtcgtacac gcgtttactt tgcggcagat 6121 gaacaaacgc tgctgaaaaa tggtaatcag accaagccga aacatgtgcc aggcacgccg 6181 tattgggtga tcaccaacac caacaccggc cgtaaatgca gcatgatcga acacatcatg 6241 cagtcgatgc aattcccggc ggaattgatt gagaaggttt gcggaactat ctaaacttaa 6301 ttaacggcac tcctcagcca agtcaaaagc ctccgaccgg aggcttttga ctacatgccc 6361 atggcgttta cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc 6421 tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca 6481 ccttgtcgcc ttgcgtataa tatttgccca tagtgaaaac gggggcgaag aagttgtcca 6541 tattggccac gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa 6601 acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat 6661 cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg 6721 aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca 6781 ccagctcacc gtctttcatt gccatacgga actccggatg agcattcatc aggcgggcaa 6841 gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg 6901 ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct 6961 caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt 7021 tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta 7081 gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccaAgcc aaataggccg 7141 t (SEQ ID NO: 34) 1 cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc actgcggaca catacaaagt 61 tacccacaga ttccgtggat aagcagagga ctaacatgtg aggcaaaaca gcagggccgc 121 gccggtggcg tttttccata ggctccgccc tcctgccaga gttcacataa acagacgctt 181 ttccggtgca tctgtgagag ccgtgaggct caaccatgaa tctgacagta cgggcgaaac 241 ccgacaggac ttaaagatcc ccgccgtttc cggcggatcg ctccctcttg cgctctcctg 301 ttccgaggct gccgtttacc ggatacctgt tccgcctttc tcccttacgg gaagtgtggc 361 gctttctcat agctcacaca ctggtatctc ggctcggtgt aggtcgttca ctccaagctg 421 ggctgtaagc aagaactccc cattcaggcc gactgctgcg ccttatccgg taactgttca 481 cttgagtgca acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg 541 gaattcgcag aggatttgtt tagctaaaca cgcggttact cttgaagtct gcgccaaagt 601 ccggctacac tggaaggaca gatttggttg ctgtagtctg cgaaagccag ttgccacggt 661 taagcagttc cccaactgac ttaaccttcg atcaaaccac ctccccaggt ggttttttcg 721 tttacagggc aaaagattac gcgcagaaaa aaaagatctc aagaagatcc tttgatcttt 781 tctactgaac cgctctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc 841 agcccaggag gaagaggaca tccgatcaaa taaaacgaaa ggctcagtcg aaagactggg 901 cctttcattt tGCTGAGGag acttagggac cctttaagac tccttattac gcagtatgtt 961 agcaaacgta gaaaatacat acataaaaat ggcaacatat aaaagaaacg caaagacacc 1021 gcggaacagg ttgatcttat cgcagtcgat actggactcg taaggtttac cagcgccaaa 1031 gacaaaaggg cgacattcaa ccgattgagg gaaagaaggt aaatattgac ggaaattatt 1141 cattaaaggt gaattatcac cgtcaccgac ttgagccatt tgggaattag agccagcaaa 1201 atcaccagta acaccattac cattaacaag gccggaaacg tcaccaatga aaccatcgat 1261 agcagcaccg taatcagtag cgacaggatc aagtttgcct ttagcgtcag actgtagcgc 1321 gttttcgtcg gcattttcgg tcgtagcccc cttattagcg tttgccatct tttcataatc 1381 aaaatcaccg aaaccagagc caccaccgga accgcctccc tcagagccgc caccctcaga 1441 accgccaccc tcagggccac caccctcaga gccgccacca gaaccaccac cagagccgcc 1501 gccagcgttg gcgggaggtt ggggcgggtc agacgattgg ccttgatatt cacaaacgaa 1561 tggatcctca ttaaagccag gatggaaagc gcagtttttg aatttaccgt tccagtaagc 1621 gtcatacatg gcttttgatg atacaggagt gtactggtaa taagttttaa cggggtcagt 1681 gccttgagta acggtgcccg tgtggacagt taatgccccc tgcctatttc ggaacctatt 1741 attctgaaac atgaaagtat taagaaactg agactcctca aaagaaggat taggattagc 1801 ggggttttgc tcagtaccag gcggataagt gccgtcgaga gggttgatat aagtatagcc 1861 cggaatgggt gtatcaccgt actcaggagg tttagtgccg ccaccctcag aaccgccacc 1921 ctcaggaccg ccaccctcag agccaccacc ctcattttca gggatagcag gtccaatagg 1981 aacccatgta ccgtaacact gagtttcgtc accagtacaa actattaacg ctgtagcatt 2041 ccacagacag ccctcatagt tagcgtaacg atctaaggtt ttgtcgtctt tccagacgtt 2101 agtaggtgaa ttttctgtat ggggttttgc taaacaactt tcaacagttt cagcggagtg 2161 agaatagaaa ggaattaact aaggaattgc gaataataat tttttcattt tttttttcct 2221 ttactgcacc tgcaggtaat gttgtcctct tgatttctgc gttcaggatt gtcctgctct 2281 ctatcactga tagggatgaa ctgttaatac aatttgcgtg ccaatttttt atcttttcga 2341 tttataaaga tctgattgag gaatcaacag caacatgcca ggatgagtta gcgaattaca 2401 ctaacaagtg gcgaatttca tcacggagcc aatatcctca gcgagtttat agaaacgcaa 2461 aaaggccgtc cgtcaggatg gccttctgct taatttgatg cctggcagtt tatggcaggc 2521 gtcctgcccg ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cccggcggat 2581 ttatcctact caggagagca ttcaccgaca aacaacagat aaaacgaaaa gcccagtctt 2641 tcgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagac 2701 cccacactac catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg 2761 accaccgcgc tactgccgcc aggcaaattc tgttttattc gaccgcttct gcgttctgat 2821 ttaatctgta tcaggctgaa aatcttctct catccgccaa aacagccagg gccctactga 2881 ctgtttatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact 2941 cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa 3001 aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg 3061 cctggctgat acgttggtcc tcgcgccagc ttaagacgct actccctaac tgctggcgga 3121 aaagatgtga cagacgcgac ggcgacaggc aaacatgctg tgcggcgctg gcgatatcaa 3181 aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca 3241 tcggtggatg aagcgactcg ttaatcactt ccatgcgccg cagtaacaat tgctcaagca 3301 gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc 3361 caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg 3421 caaatattga cggccagtta agccattcat gccagtaggc acgcggacga aagtaaaccc 3481 actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg 3541 ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc 3501 cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa 3661 aaaaattgag ataaccgttg gcctcaatcg gcgttaaacc caccaccaga tgggcattaa 3721 gcgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccac 3781 cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt 3841 actggctctt ctcgctaacc caaccaataa ccccgcttat taaaagcatt ctgtaacaaa 3901 gcgagaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag 3951 tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa 4021 gattggcgga tcctacctga cgctttttat cgcagctctc tactgtttct ccatacccgt 4081 ttttttggac gcgtacaact caagtctgac ataaatgacc gctatgagga ctgcaattac 4141 acgccagatc gttctcGCTa ccGCAaccac cggtatgaac cagattggtg cgcactatga 4201 aggccacaag atcattgaga ttggtaccgt tgaagtggtg aaccgtcgcc tgacgaacaa 4261 taacttccat gtttatctca aacccgatcg gctggtggat ccggaagcct ttggcgtaca 4321 tggtattgcc gatgaatttt tgctcgataa gcccacgttt gccgaagtag ccgatgagtt 4381 catggactat attcgcggcg cggagttggt gatccataac gcagcgttcg atatcggctt 4441 tatggactac gagttttcgt tgcttaagcg cgatattccg aagaccaata ctttctgtaa 4501 ggtcaccgat agccttgcgg tggcgaggaa aatgtttccc ggtaagcgca acagcctcga 4561 tgcgttatgt gctcgctacg aaatagataa cagtaaacga acgctgcacg gggcattact 4621 cgatgcccag atccttgcgg aagtttatct ggcgatgacc ggtggtcaaa cgtcgatggc 4631 ttttgcgatg gaaggagaaa cacaacagca acaaggtgaa gcaacaattc agcgcattgt 4741 acgtcaggca agtaagttac gcgttgtttt tgcgacagat gaagagattg cagctcatga 4801 agcccgtctc gatctggtgc agaagaaagg cggaagttgc ctctggcgag cataatttaa 4861 tatcagtaaa ccggacataa cccatgaaga aaaatcgcgc ttttttgaaa tgggcagggg 4921 gcaagtatcc cctgcttgat ggtattaaac ggcatttgcc caagggcgaa tgtctggttg 4981 agccttttgt aggtgccggg tcggtgtttc tcaacaccga cttttctcgt tatatccttg 5041 ccaatatcaa tagcgaccta atcagtctct ataacattgt gaagatgcat actgatgagt 5101 acgtacaggc cgcacgcgag ctgtttgttc ccgaaacaaa ttgcgccgag gtttactatc 5161 agttccgcga agagttcagc aaaagccagg atccgttccg tcgggcggta ctgtttttat 5221 atttgaaccg ctacggttac aacggcctgt gtcattacaa tctgcgcgat gagtttagcg 5281 tgccgttcgg ccgctacaaa aaaccctatt tcccggaagc agagttgtat cacttcgctg 5341 aaaaagcgca gaatgccttt ttctattgtg agtcttacgc cgatagcatg gcgcgcgcag 5401 atgatgcatc cgtcgtctat tgcgatccgc cttatgcacc gctgtctgcg accgccaact 5461 ttacggcgta tcacacaaac agttttacgc ttgaacaaca agcgcatctg gcggagatcg 5521 ccgaaggtct ggttgagcgc catattccag tgctggtctc caatcacgat gcgatgttaa 5581 cgcgtgagtg gtatcagcgc gcaaaattgc atgtcgtcaa agttcgacgc agtataagca 5641 gcaacggcgg cacacgtaaa aaggtggacg aactgctggc tttgtacaaa ccaggagtcg 5701 tttcgcccgc aaaaaaataa ttcagctaag acactgcact aaattaagat gaaaacaatt 5761 gaagttgatg atgaacycta cagctatatt gccagccaca ctaagcatat cggcgagagc 5821 gcatccgaca ttttacggcg tatgttgaaa ttttccgccg catcacagcc tgctgctccg 5881 gtgacgaaag aggttcgcgt tgcgtcacct gctatcgtcg aagcgaagcc ggtcaaaacg 5941 attaaagaca aggttcgcgc aatgcgtgaa cttctgcttt cggatgaata cgcagagcaa 5001 aagcgagcgg tcaatcgctt tatgctgctg ttgtctacac tatattctct tgacgcccag 6001 gcgtttgccg aagcaacgga atcgttgcac ggtcgtacac acatttactt tgcggcagat 6061 gcgtttgccg aagcaacgga atcgttgcac ggtcgtacac gcgtttactt tgcggcagat 6121 gaacaaacgc tgctgaaaaa tggtaatcag accaagccga aacatgtgcc aggcacgccg 6181 tattgggtga tcaccaacac caacaccggc cgtaaatgca gcatgatcga acacatcatg 6241 cagtcgatgc aattcccggc ggaattgatt gagaaggttt gcggaactat ctaataatac 6301 aaaaattagg aggaatttca acatgacaaa tttatctgac atcattgaaa aagaaacagg 6361 aaaacaacta gtgattcaag aatcaattct aatgttacca gaagaagtag aggaagtaat 6421 tgggaataaa ccagaaagtg atattttagt tcatactgct tatgatgaaa gtacagatga 6481 aaatgtaatg ctattaactt cagatgctcc agaatataaa ccttgggctt tagtaattca 6541 sgacagtaat ggagaaaata aaattaaaat gttataagtc gagattaagt aaaccggaat 6601 ctgaagatga ccgacgcgga atacgttcgt atccacgaaa aactggacat ctacaccttc 6661 aaaaaacagt tcttcaacaa caaaaaatct gtttctcacc gttgctacgt tctgttcgaa 6721 ctgaaacgtc gtggtgaacg tcgtgcgtgc ttctggggtt acgcggttaa caaaccgcag 6781 tctggtaccg aacgtggtat ccacgcggaa atcttctcta tccgtaaagt cgaagaatac 6841 ctgcgtgaca acccgggtca gttcaccatc aactggtact cttcttggtc tccgtgcgcg 6901 gactgcgcgg aaaaaatcct ggaatggtac aaccaggaac tgcgtggtaa cggtcacacc 6961 ctgaaaatct gggcgtgcaa actgtactac gaaaaaaacg cgcgtaacca gatcggtctg 7021 tggaacctgc gtgacaacgg tgttggtctg aacgttatgg tttctgaaca ctaccagtgc 7081 tgccgtaaaa tcttcatcca gtcttctcac aaccagctga acgaaaaccg ttggctggaa 7141 aaaaccctga aacgtgcgga aaaacgtcgt tctgaactgt ctatcatgat ccaggttaaa 7201 atcctgcaca ccaccaaatc tccggcggtt taaacttaat taacggcact cctcagccaa 7261 gtcaaaagcc tccgaccgga ggcttttgac tacatgccca tggcgtttac gccccgccct 7321 gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac 7381 aaacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat 7441 atttgcccat agtgaaaacg ggggcgaaga agttgtccat attggccacg tttaaatcaa 7501 aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca ataaaccctt 7561 tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa 7621 actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca gtttgctcat 7681 ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg tctttcattg 7741 ccatacggaa ctccggatga gcattcatca ggcgggcaag aatgtgaata aaggccggat 7801 aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg 7861 tctggttata ggtacattTa gcaactgact gaaatgcctc aaaatgttct ttacgatgcc 7921 attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta gcttcctcag 7981 ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgaccttatt tcattatggt 8041 gaaagttgga acctcttacg tgccaAgcca aataggccgt (SEQ ID NO: 35) 1 cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc gctgcggaca catacaaagt 61 tacccacaga ttccgtggat aagcaaaaaa ctaacatgtg aaacaaaaca gcagggccgc 121 gccggtggcg tttttccata ggctccgccc tcctgccaga gttcacataa acagacgctt 181 ttccggtgca tctgtgggaa ccgtgaggct caaccatgaa tctgacagta cgggcgaaac 241 ccgacaggac ttaaagatcc ccaccgtttc cggcgggtcg ctccctcttg cgctctcctg 301 ttccgaccct gccgtttacc ggatacctgc tccgcctttc tcccttacgg gaagtgtggc 361 gctttctcat agcttacaca ctggtatctc ggctcggtgt aggtcgttca ctccaagctg 421 ggctgtaagc aagaactccc cgttcagccc gactgctgcg ccttatccgg taactgttca 481 cttgagtcca acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg 541 gacttcgcag aggatttgtt tagctaaaca cgccgttgct cttgaagtat gcgccaaagt 601 ccggctacac tggaaggaca gatttggttg ctgtgctctg cgaaagccag ttaccacaat 661 taagcagttc cccaactgac ttaaccttcg atcaaaccac ctccccaggt ggttttttcg 721 tttacagggc aaaagattac gcgcagaaaa aaaagatctc aagaagatcc tttgatcttt 781 tctactgaac cactctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc 841 agcccaggag gaagaggaca tccggtcaaa taaaacgaaa ggctcagtcg aaagactggg 901 cctttcgttt tGCTGAGGag acttagggac cctttaagac tccttattac gcagtatgtt 961 agcaaacgta gaaaatacat acataaaggt ggcaacatat aaaagaaacg caaagacacc 1021 gcggaacagg ttgatcttat cgcagtcgat actgaactcg taaggttctc cagcgccaaa 1081 gacaaaaggg cgacattcaa ccgattgagg gagagaaggt aaatattgac ggaaattatt 1141 cattaaaggt gaattatcac cgtcaccgac ttgagccatt tgaaaattag agccagcaaa 1201 atcaccagta acaccattac cattaacaag gccggaaacg tcaccaatga aaccatcgat 1261 agcagcaccg taatcagtag cgacagaatc aagtttgcct ttagcgtcag actgtagcgc 1321 gttttcatcg gcattttcgg tcatagcccc cttattagcg tttgccatct tttcataatc 1381 aaaatcaccg aaaccagagc caccaccgga accgcctccc tcagagccgc caccctcaga 1441 accgccaccc tcagagccac caccctcaga gccgccacca gaaccaccac cagagccgcc 1501 gccagcattg acaggaggtt gaggcaggtc agacgattgg ccttgatatt cacaaacgaa 1561 tggatcctca ttaaagccag aatggaaagc gcagtctctg aatttaccgt tccagtaagc 1621 gtcatacatg gcttttgatg atacaggagt gtactggtaa taagttttaa cgaggtcagt 1681 gccttgagta acagtgcccg tataaacagt taatgccccc tgcctatttc ggaacctatt 1741 attctgaaac atgaaagtat taagaaactg agactcctca aaagaaggat taggattagc 1801 ggaattttgc tcagtaccag gcggataagt gccgtcgaga gggttgatat aagtatagcc 1861 cggaataggt gtatcaccgt actcaggagg tttagtaccg ccaccctcag aaccgccacc 1921 ctcagaaccg ccaccctcag agccaccacc ctcattttca aagatagcaa gcccaataga 1981 aacccatgta ccgtaacact gagtttcgtc accagtacaa actacaacgc ctgtagcatt 2041 ccacagacag ccctcatagt tagcgttacg atctaaagtt ttgtcgtctt tccagacgtt 2101 agtaaatgaa ttttctgtat gggattttgc taaacaactt tcaacagttt cagcggagtg 2161 agaatagaaa ggaacaacta aaggaattgc gaataataat tttttcattt tttttttcct 2221 ttactgcacc tgcaggtaat gttgtcctct tgatttctgc gttcaggatt gtcctgctct 2281 ctatcactga tagggatgaa ctgttaatac aatttgcgtg ccaatttttt atctttttga 2341 tttatgggga tctgattgaa gaatcaacag caacatgcca ggatgagtta gcgaattaca 2401 ctaacaagtg gcgaatttca tcacggagcc aatgtcctca gcgagtttgt agaaacgcaa 2461 aaaggccatc cgtcaggatg gccttctgct taatttgatg cctggcagtt tatggcgggc 2521 gtcctgcccg ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cccggcggat 2581 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt 2641 tcgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagac 2701 cccacactac catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg 2761 accaccgcgc tactgccgcc aggcaaattc tgttttatca gaccgcttct gcgttctgat 2821 ttaatctgta tcaggctgaa aatcttctct catccgccaa aacagccagg gccctactga 2881 ctgtttatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact 2941 cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa 3001 aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg 3061 cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga 3121 aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa 3181 aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca 3241 tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca 3301 gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc 3361 caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg 3421 caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc 3481 actggtgata ccactcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg 3541 ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc 3601 cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa 3661 aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa 3721 acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccac 3781 cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt 3841 actggctctt ctcgctaacc caaccggtaa ccccgcttat taaaagcatt ctgtaacaaa 3901 gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag 3961 tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa 4021 gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct ccatacccgt 4081 ttttttggac gcgtacaact caagtctgac ataaatgacc gctatgagca ctgcaattac 4141 acgccagatc gttctcGCTa ccGCAaccac cggtatgaac cagattggtg cgcactatga 4201 aggccacaag atcattgaga ttggtgccgt tgaagtggtg aaccgtcgcc tgacgggcaa 4261 taacttccat gtttatctca aacccgatcg gctggtggat ccggaagcct ttggcgtaca 4321 tggtattgcc gatgaatttt tgctcgataa gcccacgttt gccgaagtag ccgatgagtt 4381 catggactat attcgcggcg cggagttggt gatccataac gcagcgttcg atatcggctt 4441 tatggactac gagttttcgt tgcttaagcg cgatattccg aagaccaata ctttctgtaa 4501 ggtcaccgat agccttgcgg tggcgaggaa aatgtttccc ggtaagcgca acagcctcga 4561 tgcgttatgt gctcgctacg aaatacataa cagtaaacga acgctgcacg gggcattact 4621 cgatgcccag atccttgcgg aagtttatct ggcgatgacc ggtggtcaaa cgtcgatggc 4681 ttttgcgatg gaaggagaga cacaacagca acaaggtgaa gcaacaattc agcgcattgt 4741 acgtcaggca agtaagttac gcgttgtttt tgcgacagat gaagagattg cagctcatga 4801 agcccgtctc gatctggtgc agaagaaagg cggaagttgc ctctggcgag cataatttaa 4861 tatcagtaaa ccggacataa cccatgaaga aaaatcgcgc ttttttgaag tgggcagggg 4921 gcaagtatcc cctgcttgat gatattaaac ggcatttgcc caagggcgaa tgtctggttg 4981 agccttttgt aggtgccggg tcggtgtttc tcaacaccga cttttctcgt tatatccttg 5041 ccgatatcaa tagcgacctg atcagtctct ataacattgt gaagatgcgt actgatgagt 5101 acgtacaggc cgcacgcgag ctgtttgttc ccgaaacaaa ttgcgccgag gtttactatc 5161 agttccgcga agagttcaac aaaagccagg atccgttccg tcgggcggta ctgtttttat 5221 atttgaaccg ctacggttac aacggcctgt gtcgttacaa tctgcgcggt gagtttaacg 5281 tgccgttcgg ccgctacaaa aaaccctatt tcccggaagc agagttgtat cacttcgctg 5341 aaaaagcgca gaatgccttt ttctattgtg agtcttacgc cgatagcatg gcgcgcgcag 5401 atgatgcatc cgtcgtctat tgcgatccgc cttatgcacc gctgtctgcg accgccaact 5461 ttacggcgta tcacacaaac aghtttacgc ttgaacaaca agcgcatctg gcggagatcg 5521 ccgaaggtct ggttgagcgc catattccag tgctgatctc caatcacgat acgatgttaa 5581 cgcgtgagtg gtatcagcgc gcaaaattgc atgtcgtcaa agttcgacgc agtataagca 5641 gcaacggcgg cacacgtaaa aaggtggacg aactgctggc tttgtacaaa ccaggagtcg 5701 tttcacccgc gaaaaaataa ttcagctaag acactgcact ggattaagat gaaaacgatt 5761 gaagttgatg atgaactcta cagctatatt gccagccaca ctaagcatat cggcgagagc 5821 gcatccgaca ttttacggcg tatgttgaaa ttttccgccg catcacagcc tgctgctccg 5881 gtgacgaaag aggttcgcgt tgcgtcacct gctatcgtcg aagcgaagcc ggtcaaaacg 5941 attaaagaca aggctcgcgc aatgcgtgaa cttctgcttt cggatgaata cgcagagcaa 6001 aagcgagcgg tcaatcgctt tatgctgctg ttgtctacac tatattctct tgacgcccag 6061 gcgtttgccg aagcaacgga atcgttgcac ggtcgtacac gcgtttactt tgcggcagat 6121 gaacaaacgc tgctgaaaaa tggtaatcag accaagccga aacatgtgcc aggcacgccg 6181 tattgggtga tcaccaacac caacaccggc cgtaaatgca gcatgatcga acacatcatg 6241 cagtcgatgc aattcccggc ggaattgatt gagaaggttt gcggaactat ctaacggctg 6301 aaattaatga ggtcataccc aaatggatag ttcgtttacg cccattgaac aaatgctaaa 6361 atttcgcgcc agccgccacg aagattttcc ttatcaggag atccttctga ctcgtctttg 6421 catgcacatg caaagcaagc tgctggagaa ccgcaataaa atgctgaagg ctcagggAat 6481 taacgagacg ttgtttatgg cgttgattac gctggagtct caggaaaacc acagtattca 6541 gccttctgaa ttaagttgtg ctcttggatc atcccgtacc aacgcgacgc gtattgccga 6601 tgaactggaa aaacgcggtt ggatcgaacg tcgtgaaagc gataacgatc gccgctgcct 6661 gcatctgcaa ttaacggaaa aaggtcacga gtttttgcgc gaggttttac caccgcagca 6721 taactgcctg catcaactct ggtccgcgct cagcacaaca gaaaaagatc agctcgagca 6781 aatcacccgc aaattgctct cccgtctcga ccagatggaa caagacggtg tggttctcga 6841 agcgatgagc taataataca aaaattagga ggaatttcaa catgacaaat ttatctgaca 6901 tcattgaaaa agaaacagga aaacaactag tgattcaaga atcaattcta atgttaccag 6961 aagaagtaga ggaagtaatt gggaataaac cagaaagtga tattttagtt catactgctt 7021 atgatgaaag tacagatgaa aatgtaatgc tattaacttc agatgctcca gaatataaac 7081 cttgggcttt agtaattcaa gacagtaatg gaaaaaataa aattaaaatg ttataagtcg 7141 agattaagta aaccggaatc tgaagatgac cgacgcggaa tacgttcgta tccacgaaaa 7201 actggacatc tacaccttca aaaaacagtt cttcaacaac aaaaaatctg tttctcaccg 7261 ttgctacgtt ctgttcgaac tgaaacgtcg tggtgaacgt cgtgcgtgct tctggggtta 7321 cgcggttaac aaaccgcagt ctggtaccga acgtggtatc cacgcggaaa tcttctctat 7381 ccgtaaagtt gaagaatacc tgcgtgacaa cccgggtcag ttcaccatca actggtactc 7441 ttcttggtct ccgtgcgcgg actgcgcgga aaaaatcctg gaatggtaca accaggaact 7501 gcgtggtaac ggtcacaccc tgaaaatctg ggcgtgcaaa ctgtactacg aaaaaaacgc 7561 gcgtaaccag atcggtctgt ggaacctgcg tgacaacggt gttggtctga acgttatggt 7621 ttctgaacac taccagtgct gccgtaaaat cttcatccag tcttctcaca accagctgaa 7681 cgaaaaccgt tggctggaaa aaaccctgaa acgtgcggaa aaacgtcgtt ctgaactgtc 7741 tatcatgatc caggttaaaa tcctgcacac caccaaatct ccggcggttt aaacttaatt 7801 aacggcactc ctcagccaag tcaaaagcct ccgaccggag gcttttgact acatgcccat 7861 ggcgtttacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg 7921 ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc 7981 ttgtcgcctt acgtataata tttgcccata gtgaaaacgg gggcgaagaa gttgtccata 8041 ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac 8101 atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct 8161 tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa 8221 aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc 8281 agctcaccgt ctttcattgc catacggaac tccggatgag cattcatcag gcgggcaaga 8341 atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc 8401 gtaatatcca gccgaacggt ctggttatag gtacattTag caactgactg aaatgcccca 8461 aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc 8521 tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt 8581 gatcttattt cattatggtg aaagttggaa cctcttacgt gccaAgccaa ataggccgt

DP2 LOCUS pJC184 6537 bp DNA circular FEATURES Location/Qualifiers  modified_base 852..866   /note=“USER junction”  terminator 867..911   /note=“rrnB1 transcriptional terminator”  modified_base 919..933   /note=“USER junction”  rep_origin complement(39..777)   /dnas_title=“cloDF13”   /vntifkey=“33”   /label=cloDF13  terminator complement(7817..7852)   /note=“P14/tonB bidirectional terminator”   /note=“termination of cat transcript is slightly weaker   than in opposite direction”  modified_base 7853..7865   /note=“USER junction”  CDS complement(7866..8525)   /note=“cat (CmR)”   /note=“from pACYCDuet-1”  modified_base 7941..7941   /note=“mutation”   /note=“annotated as a G in pACYCDuet cat marker   annotation, here it is an A, but this mutation is silent   from codon GTC (Val, 25% codon usage) to GTT (Val,   21% usage), so it should not be of functional relevance”  promoter 8526..8623   /note=“cat promoter”   /note=“from pACYCDuet-1”  modified_base 8624..8639   /note=“USER junction”  CDS complement(2885..3763)   /dnas_title=“araC”   /vntifkey=“4”   /label=araC  misc_feature 2936..2936   /note=“originally an ‘a’ in annotation”  misc_feature 2975..2975   /note=“originally a t in annotation”  misc_feature 3041..3041   /note=“originally an a in annotation”  misc_feature 3245..3245   /note=“originally an c in annotation”  misc_feature 3410..3410   /note=“originally an a in annotation”  misc_feature 3569..3569   /note=“originally a g in annotation”  misc_feature 3716..3716   /note=“originally a g in annotation”  CDS complement(934..2208)   /dnas_title=“III”   /vntifkey=“4”   /label=III  misc_feature complement(1021..1066)   /note=“modified to remove internal promoter”  misc_feature 2423..2435   /note=“USER linker”  RBS complement(2209..2222)   /note=“sd8 RBS (from Ringquist and Gold Mol. Micro.   1992)”  modified_base complement(2223..2237)   /note=“USER junction”  terminator complement(2443..2867)   /note=“rrnB1 transcriptional terminator”  misc_feature 2868..2884   /note=“USER linker”  modified_base 789..806   /note=“USER Junction”  misc_difference 912..918   /note=“cloning scar”  protein_bind complement(2408..2422)   /note=“UAS II”  protein_bind complement(2386..2404)   /note=“UAS I”  prim_transcript complement(2297..2297)   /note=“Transcription Start Site”  enhancer complement(2375..2422)   /note=“Protected by pspF”  RBS complement(2406..2415)   /note=“pspF RBS”  promoter 2356..2384   /note=“pspF P1”  promoter 2375..2402   /note=“pspF P2”  promoter 2242..2269   /note=“pspF P3”  −35_signal 2242..2247   /note=“−35”  −10_signal 2264..2269   /note=“−10”  −35_signal 2356..2361   /note=“−35”  −35_signal 2375..2380   /note=“−35”  −10_signal 2379..2384   /note=“−10”  −10_signal 2397..2402   /note=“−10”  prim_transcript 2276..2276   /note=“”  prim_transcript 2390..2390   /note=“”  prim_transcript 2406..2406   /note=“”  promoter complement(2308..2324)   /note=“Sigma54 Core Promoter”  −35_signal complement(2321..2322)   /note=“−24”  −10_signal complement(2309..2310)   /note=“−12”  protein_bind complement(2326..2358)   /note=“High Affinity IHF Site”  protein_bind complement(2278..2296)   /note=“tetR binding site”  misc_feature 3861..3861   /dnas_title=“C to A ***”   /vntifkey=“21”   /label=C to A ***  misc_feature 3779..3779   /dnas_title=“A to G ***”   /vntifkey=“21”   /label=A to G ***  prim_transcript complement(3927..3927)   /note=“pC TSS”  protein_bind 3950..3966   /note=“araO1”  protein_bind 3929..3945   /note=“araO1”  protein_bind 3792..3808   /note=“araO2”  protein_bind 3971..3992   /note=“CAP”  misc_feature 4051..4051   /note=“”  misc_feature 4037..4037   /note=“”  prim_transcript 4075..4075   /note=“pBAD TSS”  protein_bind 4003..4019   /note=“ara I1”  protein_bind 4024..4040   /note=“araI2”  −10_signal 4061..4066   /note=“−10”  −35_signal 4037..4042   /note=“−35”  promoter 3971..4100   /dnas_title=“pBAD”   /vntifkey=“30”   /label=pBAD  RBS 4104..4123   /note=“Native dnaQ RBS”  CDS 4124..4855   /dnas_title=“dnaQ926”   /vntifkey=“4”   /label=dnaQ926  conflict 4157..4159   /note=“D12A”  conflict 4163..4165   /note=“E14A”  RBS 4865..4883   /note=“Modified mutS RBS”  modified_base 4865..4878   /note=“USER Junction”  CDS 4884..5720   /note=“dam (wt)”  CDS 5749..6294   /note=“seqA (wt)”  RBS 5729..5748   /note=“seqA Native RBS”  CDS 6323..6853   /note=“emrR (wt)”  RBS 6303..6322   /note=“native emrR RBS”  modified_base 6275..6292   /note=“USER Junction”  CDS 6882..7136   /note=“PBS2 UGI”  RBS 6861..6881   /note=“native UGI RBS”  modified_base 6858..6877   /note=“USER Junction”  RBS 7145..7165   /note=“dnaE RBS”  modified_base 7141..7159   /note=“USER Junction”  CDS 7166..7792   /note=“pmCDA1 (opt)”  modified_base 7793..7809   /note=“USER junction”  source 1..5702

DP3 LOCUS pJC184 6537 bp DNA circular FEATURES Location/Qualifiers  modified_base 852..866   /note=“USER junction”  terminator 867..911   /note=“rrnB1 transcriptional terminator”  modified_base 919..933   /note=“USER junction”  rep_origin complement(39..777)   /dnas_title=“cloDF13”   /vntifkey=“33”   /label=cloDF13  terminator complement(5745..5780)   /note=“P14/tonB bidirectional terminator”   /note=“termination of cat transcript is slightly weaker   than in opposite direction”  modified_base 5781..5793   /note=“USER junction”  CDS complement(5794..6453)   /note=“cat (CmR)”   /note=“from pACYCDuet-1”  modified_base 5869..5869   /note=“mutation”   /note=“annotated as a G in pACYCDuet cat marker   annotation, here it is an A, but this mutation is silent   from codon GTC (Val, 25% codon usage) to GTT (Val,   21% usage), so it should not be of functional relevance”  promoter 6454..6551   /note=“cat promoter”   /note=“from pACYCDuet-1”  modified_base 6552..6567   /note=“USER junction”  CDS complement(2885..3763)   /dnas_title=“araC”   /vntifkey=“4”   /label=araC  misc_feature 2936..2936   /note=“originally an ‘a’ in annotation”  misc_feature 2975..2975   /note=“originally a t in annotation”  misc_feature 3041..3041   /note=“originally an a in annotation”  misc_feature 3245..3245   /note=“originally an c in annotation”  misc_feature 3410..3410   /note=“originally an a in annotation”  misc_feature 3569..3569   /note=“originally a g in annotation”  misc_feature 3716..3716   /note=“originally a g in annotation”  CDS complement(934..2208)   /dnas_title=“III”   /vntifkey=“4”   /label=III  misc_feature complement(1021..1066)   /note=“modified to remove internal promoter”  misc_feature 2423..2435   /note=“USER linker”  RBS complement(2209..2222)   /note=“sd8 RBS (from Ringquist and Gold Mol. Micro.   1992)”  modified_base complement(2223..2237)   /note=“USER junction”  terminator complement(2443..2867)   /note=“rrnB1 transcriptional terminator”  misc_feature 2868..2884   /note=“USER linker”  modified_base 789..806   /note=“USER Junction”  misc_difference 912..918   /note=“cloning scar”  protein_bind complement(2408..2422)   /note=“UAS II”  protein_bind complement(2386..2404)   /note=“UAS I”  prim_transcript complement(2297..2297)   /note=“Transcription Start Site”  enhancer complement(2375..2422)   /note=“Protected by pspF”  RBS complement(2406..2415)   /note=“pspF RBS”  promoter 2356..2384   /note=“pspF P1”  promoter 2375..2402   /note=“pspF P2”  promoter 2242..2269   /note=“pspF P3”  −35_signal 2242..2247   /note=“−35”  −10_signal 2264..2269   /note=“−10”  −35_signal 2356..2361   /note=“−35”  −35_signal 2375..2380   /note=“−35”  −10_signal 2379..2384   /note=“−10”  −10_signal 2397..2402   /note=“−10”  prim_transcript 2276..2276   /note=“”  prim_transcript 2390..2390   /note=“”  prim_transcript 2406..2406   /note=“”  promoter complement(2308..2324)   /note=“Sigma54 Core Promoter”  −35_signal complement(2321..2322)   /note=“−24”  −10_signal complement(2309..2310)   /note=“−12”  protein_bind complement(2326..2358)   /note=“High Affinity IHF Site”  protein_bind complement(2278..2296)   /note=“tetR binding site”  misc_feature 3861..3861   /dnas_title=“C to A ***”   /vntifkey=“21”   /label=C to A ***  misc_feature 3779..3779   /dnas_title=“A to G ***”   /vntifkey=“21”   /label=A to G ***  prim_transcript complement(3927..3927)   /note=“pC TSS”  protein_bind 3950..3966   /note=“araO1”  protein_bind 3929..3945   /note=“araO1”  protein_bind 3792..3808   /note=“araO2”  protein_bind 3971..3992   /note=“CAP”  misc_feature 4051..4051   /note=“”  misc_feature 4037..4037   /note=“”  prim_transcript 4075..4075   /note=“pBAD TSS”  protein_bind 4003..4019   /note=“ara I1”  protein_bind 4024..4040   /note=“araI2”  −10_signal 4061..4066   /note=“−10”  −35_signal 4037..4042   /note=“−35”  promoter 3971..4100   /dnas_title=“pBAD”   /vntifkey=“30”   /label=pBAD  RBS 4104..4123   /note=“Native dnaQ RBS”  CDS 4124..4855   /dnas_title=“dnaQ926”   /vntifkey=“4”   /label=dnaQ926  conflict 4157..4159   /note=“D12A”  conflict 4163..4165   /note=“E14A”  RBS 4865..4883   /note=“Modified mutS RBS”  modified_base 4865..4878   /note=“USER Junction”  CDS 4884..5720   /note=“dam (wt)”  modified_base 5721..5737   /note=“USER junction”  source 1..6567

DP4 LOCUS pJC184 6537 bp DNA circular FEATURES Location/Qualifiers  modified_base 852..866   /note=“USER junction”  terminator 867..911   /note=“rrnB1 transcriptional terminator”  modified_base 919..933   /note=“USER junction”  rep_origin complement(39..777)   /dnas_title=“cloDF13”   /vntifkey=“33”   /label=cloDF13  terminator complement(6319..6354)   /note=“P14/tonB bidirectional terminator”   /note=“termination of cat transcript is slightly weaker than in opposite direction”  modified_base 6355..6367   /note=“USER junction”  CDS complement(6368..7027)   /note=“cat (CmR)”   /note=“from pACYCDuet-1”  modified_base 6443..6443   /note=“mutation”   /note=“annotated as a G in pACYCDuet cat marker   annotation, here it is an A, but this mutation is silent   from codon GTC (Val, 25% codon usage) to GTT (Val,   21% usage), so it should not be of functional relevance”  promoter 7028..7125   /note=“cat promoter”   /note=“from pACYCDuet-1”  modified_base 7126..7141   /note=“USER junction”  CDS complement(2885..3763)   /dnas_title=“araC”   /vntifkey=“4”   /label=araC  misc_feature 2936..2936   /note=“originally an ‘a’ in annotation”  misc_feature 2975..2975   /note=“originally a t in annotation”  misc_feature 3041..3041   /note=“originally an a in annotation”  misc_feature 3245..3245   /note=“originally an c in annotation”  misc_feature 3410..3410   /note=“originally an a in annotation”  misc_feature 3569..3569   /note=“originally a g in annotation”  misc_feature 3716..3716   /note=“originally a g in annotation”  CDS complement(934..2208)   /dnas_title=“III”   /vntifkey=“4”   /label=III  misc_feature complement(1021..1066)   /note=“modified to remove internal promoter”  misc_feature 2423..2435   /note=“USER linker”  RBS complement(2209..2222)   /note=“sd8 RBS (from Ringquist and Gold Mol. Micro.   1992)”  modified_base complement(2223..2237)   /note=“USER junction”  terminator complement(2443..2867)   /note=“rrnB1 transcriptional terminator”  misc_feature 2868..2884   /note=“USER linker”  modified_base 789..806   /note=“USER Junction”  misc_difference 912..918   /note=“cloning scar”  protein_bind complement(2408..2422)   /note=“UAS II”  protein_bind complement(2386..2404)   /note=“UAS I”  prim_transcript complement(2297..2297)   /note=“Transcription Start Site”  enhancer complement(2375..2422)   /note=“Protected by pspF”  RBS complement(2406..2415)   /note=“pspF RBS”  promoter 2356..2384   /note=“pspF P1”  promoter 2375..2402   /note=“pspF P2”  promoter 2242..2269   /note=“pspF P3”  −35_signal 2242..2247   /note=“−35”  −10_signal 2264..2269   /note=“−10”  −35_signal 2356..2361   /note=“−35”  −35_signal 2375..2380   /note=“−35”  −10_signal 2379..2384   /note=“−10”  −10_signal 2397..2402   /note=“−10”  prim_transcript 2276..2276   /note=“”  prim_transcript 2390..2390   /note=“”  prim_transcript 2406..2406   /note=“”  promoter complement(2308..2324)   /note=“Sigma54 Core Promoter”  −35_signal complement(2321..2322)   /note=“−24”  −10_signal complement(2309..2310)   /note=“−12”  protein_bind complement(2326..2358)   /note=“High Affinity IHF Site”  protein_bind complement(2278..2296)   /note=“tetR binding site”  misc_feature 3861..3861   /dnas_title=“C to A ***”   /vntifkey=“21”   /label=C to A ***  misc_feature 3779..3779   /dnas_title=“A to G ***”   /vntifkey=“21”   /label=A to G ***  prim_transcript complement(3927..3927)   /note=“pC TSS”  protein_bind 3950..3966   /note=“araO1”  protein_bind 3929..3945   /note=“araO1”  protein_bind 3792..3808   /note=“araO2”  protein_bind 3971..3992   /note=“CAP”  misc_feature 4051..4051   /note=“”  misc_feature 4037..4037   /note=“”  prim_transcript 4075..4075   /note=“pBAD TSS”  protein_bind 4003..4019   /note=“ara I1”  protein_bind 4024..4040   /note=“araI2”  −10_signal 4061..4066   /note=“−10”  −35_signal 4037..4042   /note=“−35”  promoter 3971..4100   /dnas_title=“pBAD”   /vntifkey=“30”   /label=pBAD  RBS 4104..4123   /note=“Native dnaQ RBS”  CDS 4124..4855   /dnas_title=“dnaQ926”   /vntifkey=“4”   /label=dnaQ926  conflict 4157..4159   /note=“D12A”  conflict 4163..4165   /note=“E14A”  RBS 4865..4883   /note=“Modified mutS RBS”  modified_base 4865..4878   /note=“USER Junction”  CDS 4884..5720   /note=“dam (wt)”  CDS 5749..6294   /note=“seqA (wt)”  RBS 5729..5748   /note=“seqA Native RBS”  modified_base 6275..6311   /note=“USER junction”  source 1..7141

DP5 LOCUS pJC184 6537 bp DNA circular FEATURES Location/Qualifiers  modified_base 852..866   /note=“USER junction”  terminator 867..911   /note=“rrnB1 transcriptional terminator”  modified_base 919..933   /note=“USER junction”  rep_origin complement(39..777)   /dnas_title=“cloDF13”   /vntifkey=“33”   /label=cloDF13  terminator complement(7258..7293)   /note=“P14/tonB bidirectional terminator”   /note=“termination of cat transcript is slightly weaker   than in opposite direction”  modified_base 7294..7306   /note=“USER junction”  CDS complement(7307..7966)   /note=“cat (CmR)”   /note=“from pACYCDuet-1”  modified_base 7382..7382   /note=“mutation”   /note=“annotated as a G in pACYCDuet cat marker   annotation, here it is an A, but this mutation is silent   from codon GTC (Val, 25% codon usage) to GTT (Val,   21% usage), so it should not be of functional relevance”  promoter 7967..8064   /note=“cat promoter”   /note=“from pACYCDuet-1”  modified_base 8065..8080   /note=“USER junction”  CDS complement(2885..3763)   /dnas_title=“araC”   /vntifkey=“4”   /label=araC  misc_feature 2936..2936   /note=“originally an ‘a’ in annotation”  misc_feature 2975..2975   /note=“originally a t in annotation”  misc_feature 3041..3041   /note=“originally an a in annotation”  misc_feature 3245..3245   /note=“originally an c in annotation”  misc_feature 3410..3410   /note=“originally an a in annotation”  misc_feature 3569..3569   /note=“originally a g in annotation”  misc_feature 3716..3716   /note=“originally a g in annotation”  CDS complement(934..2208)   /dnas_title=“III”   /vntifkey=“4”   /label=III  misc_feature complement(1021..1066)   /note=“modified to remove internal promoter”  misc_feature 2423..2435   /note=“USER linker”  RBS complement(2209..2222)   /note=“sd8 RBS (from Ringquist and Gold Mol. Micro.   1992)”  modified_base complement(2223..2237)   /note=“USER junction”  terminator complement(2443..2867)   /note=“rrnB1 transcriptional terminator”  misc_feature 2868..2884   /note=“USER linker”  modified_base 789..806   /note=“USER Junction”  misc_difference 912..918   /note=“cloning scar”  protein_bind complement(2408..2422)   /note=“UAS II”  protein_bind complement(2386..2404)   /note=“UAS I”  prim_transcript complement(2297..2297)   /note=“Transcription Start Site”  enhancer complement(2375..2422)   /note=“Protected by pspF”  RBS complement(2406..2415)   /note=“pspF RBS”  promoter 2356..2384   /note=“pspF P1”  promoter 2375..2402   /note=“pspF P2”  promoter 2242..2269   /note=“pspF P3”  −35_signal 2242..2247   /note=“−35”  −10_signal 2264..2269   /note=“−10”  −35_signal 2356..2361   /note=“−35”  −35_signal 2375..2380   /note=“−35”  −10_signal 2379..2384   /note=“−10”  −10_signal 2397..2402   /note=“−10”  prim_transcript 2276..2276   /note=“”  prim_transcript 2390..2390   /note=“”  prim_transcript 2406..2406   /note=“”  promoter complement(2308..2324)   /note=“Sigma54 Core Promoter”  −35_signal_ complement(2321..2322)   /note=“−24”  −10_signal complement(2309..2310)   /note=“−12”  protein_bind complement(2326..2358)   /note=“High Affinity IHF Site”  protein_bind complement(2278..2296)   /note=“tetR binding site”  misc_feature 3861..3861   /dnas_title=“C to A ***”   /vntifkey=“21”   /label=C to A ***  misc_feature 3779..3779   /dnas_title=“A to G ***”   /vntifkey=“21”   /label=A to G ***  prim_transcript complement(3927..3927)   /note=“pC TSS”  protein_bind 3950..3966   /note=“araO1”  protein_bind 3929..3945   /note=“araO1”  protein_bind 3792..3808   /note=“araO2”  protein_bind 3971..3992   /note=“CAP”  misc_feature 4051..4051   /note=“”  misc_feature 4037..4037   /note=“”  prim_transcript 4075..4075   /note=“pBAD TSS”  protein_bind 4003..4019   /note=“ara I1”  protein_bind 4024..4040   /note=“araI2”  −10_signal 4061..4066   /note=“−10”  −35_signal 4037..4042   /note=“−35”  promoter 3971..4100   /dnas_title=“pBAD”   /vntifkey=“30”   /label=pBAD  RBS 4104..4123   /note=“Native dnaQ RBS”  CDS 4124..4855   /dnas_title=“dnaQ926”   /vntifkey=“4”   /label=dnaQ926  conflict 4157..4159   /note=“D12A”  conflict 4163..4165   /note=“E14A”  RBS 4865..4883   /note=“Modified mutS RBS”  modified_base 4865..4878   /note=“USER Junction”  CDS 4884..5720   /note=“dam (wt)”  CDS 5749..6294   /note=“seqA (wt)”  RBS 5729..5748   /note=“seqA Native RBS”  modified_base 6275..6292   /note=“USER Junction”  CDS 6323..6577   /note=“PBS2 UGI”  RBS 6302..6322   /note=“Native UGI RBS”  modified_base 6299..6318   /note=“USER Junction”  RBS 6586..6606   /note=“dnaE RBS”  modified_base 6582..6600   /note=“USER Junction”  CDS 6607..7233   /note=“pmCDA1 (opt)”  modified_base 7234..7250   /note=“USER junction”  source 1..8080

DP6 LOCUS pJC184 6537 bp DNA circular FEATURES Location/Qualifiers  modified_base 852..866   /note=“USER junction”  terminator 867..911   /note=“rrnB1 transcriptional terminator”  modified_base 919..933   /note=“USER junction”  rep_origin complement(39..777)   /dnas_title=“cloDF13”   /vntifkey=“33”   /label=cloDF13  terminator complement(7817..7852)   /note=“P14/tonB bidirectional terminator”   /note=“termination of cat transcript is slightly weaker   than in opposite direction”  modified_base 7853:7865   /note=“USER junction”  CDS complement(7866..8525)   /note=“cat (CmR)”   /note=“from pACYCDuet-1”  modified_base 7941..7941   /note=“mutation”   /note=“annotated as a G in pACYCDuet cat marker   annotation, here it is an A, but this mutation is silent   from codon GTC (Val, 25% codon usage) to GTT (Val,   21% usage), so it should not be of functional relevance”  promoter 8526..8623   /note=“cat promoter”   /note=“from pACYCDuet-1”  modified_base 8624..8639   /note=“USER junction”  CDS complement(2885..3763)   /dnas_title=“araC”   /vntifkey=“4”   /label=araC  misc_feature 2936..2936   /note=“originally an ‘a’ in annotation”  misc_feature 2975..2975   /note=“originally a t in annotation”  misc_feature 3041..3041   /note=“originally an a in annotation”  misc_feature 3245..3245   /note=“originally an c in annotation”  misc_feature 3410..3410   /note=“originally an a in annotation”  misc_feature 3569..3569   /note=“originally a g in annotation”  misc_feature 3716..3716   /note=“originally a g in annotation”  CDS complement(934..2208)   /dnas_title=“III”   /vntifkey=“4”   /label=III  misc_feature complement(1021.. 1066)   /note=“modified to remove internal promoter”  misc_feature 2423..2435   /note=“USER linker”  RBS complement(2209..2222)   /note=“sd8 RBS (from Ringquist and Gold Mol. Micro.   1992)”  modified_base complement(2223..2237)   /note=“USER junction”  terminator complement(2443..2867)   /note=“rrnBI transcriptional terminator”  misc_feature 2868..2884   /note=“USER linker”  modified_base 789..806   /note=“USER Junction”  misc_difference 912..918   /note=“cloning scar”  protein_bind complement(2408..2422)   /note=“UAS II”  protein_bind complement(2386..2404)   /note=“UAS I”  prim_transcript complement(2297..2297)   /note=“Transeription Start Site”  enhancer complement(2375..2422)   /note=“Protected by pspF”  RBS complement(2406..2415)   /note=“pspF RBS”  promoter 2356..2384   /note=“pspF P1”  promoter 2375..2402   /note=“pspF P2”  promoter 2242..2269   /note=“pspF P3”  −35_signal 2242..2247   /note=“−3”  −10_signal 2264..2269   /note=“−10”  −35_signal 2356..2361   /note=“−35”  −35_signal 2375..2380   /note=“−35”  −10_signal 2379..2384   /note=“−10”  −10_signal 2197..2402   /note=“−10”  prim_transcript 2276..2276   /note=“”  prim_transcript 2390..2390   /note=“”  prim_transcript 2406..2406   /note=“”  promoter complement(2308..2324)   /note=“Sigma54 Core Promoter”  −35_signal_ complement(2321..2322)   /note=“−24”  −10_signal complement(2309..2310)   /note=“−12”  protein_bind complement(2326..2358)   /note=“High Affinity IHF Site”  protein_bind complement(2278..2296)   /note=“tetR binding site”  misc_feature 3861..3861   /dnas_title=“C to A ***”   /vntifkey=“21”   /label=C to A ***  misc_feature 3779..3779   /dnas_title=“A to G ***”   /vntifkey=“21”   /label=A to G ***  prim_transcript complement(3927..3927)   /note=“pC TSS”  protein_bind 3950..3966   /note=“araO1”  protein_bind 3929..3945   /note=“araO1”  protein_bind 3792..3808   /note=“araO2”  protein_bind 3971..3992   /note=“CAP”  misc_feature 4051..4051   /note=“”  misc_feature 4037..4037   /note=“”  prim_transcript 4075..4075   /note=“pBAD TSS”  protein_bind 4003..4019   /note=“ara I1”  protein_bind 4024..4040   /note=“araI2”  −10_signal 4061..4066   /note=“−10”  −35_signal 4037..4042   /note=“−35”  promoter 3971..4100   /dnas_title=“pBAD”   /vntifkey=“30”   /label=pBAD  RBS 4104..4123   /note=“Native dnaQ RBS”  CDS 4124..4855   /dnas_title=“dnaQ926”   /vntifkey=“4”   /label=dnaQ926  conflict 4157..4159   /note=“D12A”  conflict 4163..4165   /note=“E14A”  RBS 4865..4883   /note=“Modified mutS RBS”  modified_base 4865..4878   /note=“USER Junction”  CDS 4884..5720   /note=“−35”  CDS 5749..6294   /note=“seqA (wt)”  RBS 5729..5748   /note=“seqA Native RBS”  CDS 6323..6853   /note=“emrR (wt)”  RBS 6303..6322   /note=“native emrR RBS”  modified_base 6275..6292   /note=“USER Junction”  CDS 6882..7136   /note=“PBS2 UGI”  RBS 6861..6881   /note=“Native UGI RBS”  modified_base 6858..6877   /note=“USER Junction”  RBS 7145..7165   /note=“dnaE RBS”  modified_base 7141..7159   /note=“USER Junction”  CDS 7166..7792   /note=“pmCDA1 (opt)”  modified_base 7793..7809   /note=“USER junction”  source 1..8639

Exemplary Sequences

Non-limiting sequences of exemplary encoding gene products that increase the mutation rate in a host cell, e.g., in a bacterial host cell, are provided below. Those of ordinary skill in the art will understand that other useful sequences for each of the gene products provided exist, e.g., sequences having one or more point mutations that do not affect the sequence or function of the encoded RNA or protein.

araC (SEQ ID NO: 33) atga caacttgacg gctacatcat tcactttttc ttcacaacca gcacggaact cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttgatcc tcgcgccagc ttaagacgct aatccctaac tgctaggga aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc caaacaggtc gctgaaatgc ggctgatgcg cr tcatccg gcgaaagaac cccgtattgg caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc actgatgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccccgattt ttcaccaccc cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cgatcgataa aaaaatcgag ataaccgtta gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa acgagtatcc cggcagcagg ggatcatttt gcgcttcagc cat dnaQ926 (SEQ ID NO: 37) atgag cactgcaatt acacgccaga tcgttctcGC TaccGCAacc accggtatga accagattgg tgcgcactat gaaggccaca agatcattga gattggtgcc gttgaagtgg tgaaccgtcg cctgacgggc aataacttcc atgtttatct caaacccgat cggctgatgg atccggaagc ctttggcgta catggtattg ccgatgaatt tttgctcgat aagcccacgt ttgccgaagt agccgatgag ttcatggact atattcgcgg cgcggagttg gtgatccata acgcagcatt cgatatcggc tttatggact acgagttttc gttgcttaag cgcgatattc cgaaaaccaa tactttctgt aagatcaccg atagccttgc ggtggcgagg aaaatgtttc ccggtaagcg caacagcctc gatgcgttat gtgctcgctd cgaaatagat aacagtaaac gaacgctgca cggggcatta ctcgatgccc agatccttgc ggaagtttat ctggcgatga ccggtagtca aacgtcgatg gcttttgcga tggaaagaga gacacaacag caacaaggtg aagcaacaat tcagcgcatt gtacgtcagg caagtaagtt acgcgttgtt tttgcgacag atgaagagat tgcagctcat gaagcccgtc tcgatctggt gcagagaaa ggcggaagtt gcctctggcg agcataa dam (SEQ ID NO: 38) atgaa gaaaaatcgc gcttttttga agtgggcagg gggcaagtat cccctgcttg atgatattaa acggcatttg cccaagggcg aatgtctggt tgagcctttt gtaggtgccg gatcggtgtt tctcaacacc gacttttctc gttatatcct tgccgatatc aatagcgacc tgatcagtct ctataacatt gtgaagatgc gtactgatga gtacgtacag gccgcacgcg agctgtttgt tcccgaaaca aattgcgccg aggtttacta tcagttccgc gaagagttca acaaaagcca ggatccgttc cgtcgggcgg tactgttttt atatttgaac cgctacaatt acaacggcct gtgtcgttac aatctgcgcg gtgagtttaa cgtgccgttc ggccactaca aaaaacccta tttcccggaa gcagagttgt atcacttcgc tgaaaaagcg cagaatgcct ttttctattg tgagtcttac gccgatagca tggcgcgcgc agatgatgca tccgtcgtct attgcgatcc gccttatgca ccgctgtctg cgaccgccaa ctttacggcg tatcacacaa acagttttac acttgaacaa caagcgcatc tggcggagat cgccgaaggt ctgattgagc gccatattcc agtgctgatc tccaatcacg atacgatgtt aacgcgtgag tggtatcagc gcgcaaaatt gcatatcgtc aaagttcgac gcagtataaa cagcaacggc ggcacacgta aaaaggtgga cgaactgctg gctttgtaca aaccaggagt cgtttcaccc gcgaaaaaat aa seqA (SEQ ID NO: 39) atgaaaacga ttaaagttga tgatgaactc tacagctata ttaccagcca cactaagcat atcggcgaga gcgcatccga cattttacgg cgtatgttga aattttccgc cgcatcacag cctgctgctc cggtgacgaa agaggttcgc gttgcgtcac ctgctatcgt cgaagcgaag ccggtcaaaa cgattaaaga caaggttcgc gcaatgcgtg aacttctgct ttcggatgaa tacgcagagc aaaagcgagc ggtcaatcgc tttatgctgc tgttgtctac actatattct cttgacgccc aggcgtttgc cgaagcaacg gaatcgttgc acggtcgtac acgcgtttac tttgcggcag atgaacaaac gctgctgaaa aatagtaatc agaccaagcc gaaacatgtg ccaggcacgc cgtattaggt gatcaccaac accaacaccg gccgtaaatg cagcatgatc gaacacatca tgcagtcgat gcaattcccg gcggaattga ttgagaaggt ttgcggaact atctaa emrR (SEQ ID NO: 40) atggat agttcgttta cgcccattga acaaatgcta aaatttcgcg ccagccgcca caaagacttt ccttatcagg agatccttct gactcgtctt tgcatgcaca tgcaaagcaa gctgctggag aaccgcaata aaatactgaa ggctcagggA attaacgaga cgttatttat ggcgttgatt acgctggagt ctcaggaaaa ccacagtatt cagccttctg aattaagttg tgctcttgga tcatcccgta ccaacgcgac gcgtattgcc gatgaactgg aaaaacgcgg ttggatcgaa cgtcgtgaaa gcgataacga tcgccgctgc ctgcatctgc aattaacgga aaaaggtcac aagtttttgc gcgaggtttt accaccgcag cataactgcc tgcatcaact ctggtccgcg ctcagcacaa cagaaaaaga tcagctcgag caaatcaccc gcaaattgct ctcccgtctc gaccagatgg aacaagacgg tgtggttctc gaagcgatga gctaa UGI (SEQ ID NO: 41) atgacaa atttatctga catcattgaa aaagaaacag gaaaattaact agtgattcaa gaatcaattc taatgttacc agaagaagta gaggaagtaa ttgggaataa accagaaagt gatattttag ttcatactgc ttatgatgaa agtacagatg aaaatgtaat gctattaact tcagatgctc cagaatataa accttgggct ttagtaattc aagacagtaa tggagaaaat aaaattaaaa tgttataa CDA1 (SEQ ID NO: 42) atg accgacgcgg aatacgttcg tatccacgaa aaactggaca tctacacctt caaaaaacag ttcttcaaca acaaaaaatc tctttctcac cgttgctacg ttctgttcga actgaaacgt cgtgatgaac gtcgtgcgtg cttctgaggt tacgcgatta acaaaccgca gtctgatacc gaacgtggta tccacgcgga aatcttctct atccgtaaag ttgaagaata cctgcgtgac aacccgggtc agttcaccat caactggtac tcttcttaat ctccgtgcgc gaactgcgcg gaaaaaatcc tggaatggta caaccaggaa ctgcgtggta acggtcacac cctgaaaatc tgggcgtgca aactgtacta cgaaaaaaac gcgcgtaacc agatcggtct gtggaacctg cgtgacaacg gtattggtct gaacgttatg gtttctgaac actaccagtg ctgccgtaaa atcttcatcc agtcttctca caaccagctg aacgaaaacc gttggctgga aaaaaccctg aaacgtgcgg aaaaacgtcg ttctgaacta tctatcatga tccaggttaa aatcctgcac accaccaaat ctccggcggt ttaa Drift Promoter (SEQ ID NO: 124) aat gttgtcctct tgatttctgc gttcaggatt gtcctgctct ctatcactga tagggatgaa ctgttaatac aatttgcgtg ccaatttttt atctttttga tttataaaga tctgattgaa gaatcaacag caacatgcca ggatgagtta gcgaattaca ctaacaagtg gcgaatttca tc

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All publications, patents, patent applications, publication, and database entries (e.g., sequence database entries) mentioned herein, e.g., in the Background, Summary, Detailed Description, Examples, and/or References sections, are hereby incorporated by reference in their entirety as if each individual publication, patent, patent application, publication, and database entry was specifically and individually incorporated herein by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context. The disclosure of a group that includes “or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

It is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitation, element, clause, or descriptive term, from one or more of the claims or from one or more relevant portion of the description, is introduced into another claim. For example, a claim that is dependent on another claim can be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, if any, are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, it is to be understood that every possible subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where an embodiment, product, or method is referred to as comprising particular elements, features, or steps, embodiments, products, or methods that consist, or consist essentially of, such elements, features, or steps, are provided as well. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in some embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, the values in each range have not been individually spelled out herein, but it will be understood that each of these values is provided herein and may be specifically claimed or disclaimed. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

Claims

1-125. (canceled)

126. An expression construct for modulating the mutation rate of nucleic acids in a bacterial cell, the construct comprising:

(i) a drift promoter; and
(ii) a nucleic acid sequence encoding one or more gene products selected from a gene product that disrupts a proofreading pathway, a translesion synthesis pathway, a methyl-directed mismatch repair pathway, a base excision repair pathway, or a base selection pathway of the bacterial cell, or any combination thereof,
wherein the nucleic acid sequence encoding the one or more gene products is under the control of the drift promoter.

127. The expression construct of claim 126, wherein the gene product that disrupts a proofreading pathway is a dnaQ926, BRM1, BR11, BR1, BR6, or BR13 gene product.

128. The expression construct of claim 126, wherein the gene product that disrupts a translesion synthesis pathway is an umuD′, umuC, recA, dinB, or polB gene product.

129. The expression construct of claim 126, wherein the gene product that disrupts a methyl-directed mismatch repair pathway is a mutS, mutL, mutH, dam, or seqA gene product.

130. The expression construct of claim 126, wherein the gene product that disrupts a base excision repair pathway is a ugi, AID, APOBEC, CDA, MAG, or AAG gene product.

131. The expression construct of claim 126, wherein the gene product that disrupts a base selection pathway is a dnaE74, dnaE486, dnaE1026, dnaX36, dnaX2016, emrR, nrdAB, nrdA(H59A)B, nrdA(A65V)B, nrdA(A301V)B, nrdAB(P334L), or nrdEF gene product.

132. The expression construct of claim 126, wherein the expression construct further comprises a nucleic acid sequence encoding a rsmE, cchA, yffI, or yfjY gene product.

133. The expression construct of claim 126, wherein the expression construct comprises a nucleic acid sequence encoding a dnaQ926 gene product.

134. The expression construct of claim 126, wherein the expression construct comprises a nucleic acid sequence encoding a dnaE74, a dnaE486, a dnaE1026, a dnaX36, a dnaX2016, a rpsD12, a rpsD14, a rpsD16, a polB, a polB(D156A), a MAG1, a AAG(Y127I-H136L), and/or a Δ80-AAG(Y127I-H136L) gene product.

135. The expression construct of claim 126, wherein the one or more gene product is dnaQ926, umuD′, umuC, recA730, dam, seqA, emrR, PBS2, UGI, or CDA1, or any combination thereof.

136. The expression construct of claim 126, wherein the expression construct comprises a nucleic acid sequence encoding a: dnaQ926 gene product, a dam gene product, a seqA gene product, an emrR gene product, a ugi gene product, and a CDA1 gene product.

137. The expression construct of claim 126 comprising the sequence set forth in any one of SEQ ID NO: 27, 28, 29, 33, 34, and 35.

138. The expression construct of claim 126, wherein the drift promoter comprises the sequence set forth in SEQ ID NO: 124.

139. The expression construct of claim 126, wherein the expression construct comprises a nucleic acid sequence encoding an arabinose operon regulatory protein.

140. The expression construct of claim 139, wherein the nucleic acid sequence encoding an arabinose operon regulatory protein comprises the sequence set forth in SEQ ID NO: 36.

141. A cell comprising the expression construct of claim 126.

142. A plasmid comprising the expression construct of claim 126.

143. The plasmid of claim 142, wherein the plasmid comprises a bacterial origin of replication.

144. The plasmid of claim 143, wherein the origin of replication is a cloDF13 origin of replication.

145. The plasmid of claim 142 further comprising an expression construct comprising a nucleic acid sequence encoding pIII protein.

Patent History
Publication number: 20220267754
Type: Application
Filed: Jan 21, 2022
Publication Date: Aug 25, 2022
Applicant: President and Fellows of Harvard College (Cambridge, MA)
Inventors: Ahmed Hussein Badran (Cambridge, MA), David R. Liu (Cambridge, MA)
Application Number: 17/581,235
Classifications
International Classification: C12N 15/10 (20060101); C12N 9/12 (20060101); C12N 9/48 (20060101); C12N 15/70 (20060101);