Abstract: The invention relates to a fast method of transforming competent cells, comprising: a. mixing a plasmid DNA with the competent cells suspending within ionic solution to form a mixture; b. plating the mixture on a warm selective medium; and c. culturing the mixture on the medium; wherein the ionic solution comprises divalent cation selected from the group consisting of Ca2+, Mg2+, Mn2+, Zn2+, Cu2+, Cd2+, Fe2+, Sr2+ and Co2+, and provided that the ionic solution does not include Ca2+ alone.

Abstract: Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Abstract: The present invention is directed to mutants of lysine decarboxylase, nucleic acids encoding the mutants, and vaccines comprising the mutants for inhibiting and reducing the development of periodontal diseases, including gingivitis and chronic periodontitis. The vaccine composition comprises a recombinant lysine decarboxylase mutant which is based on a native version of the enzyme from E. corrodens and induces production of antibodies that inhibit the activity of the lysine decarboxylase enzyme in the oral cavity. The recombinant lysine decarboxylase mutant, in one version, comprises a mutation at residue 365 or at other locations within the active site, and in a preferred embodiment is produced from E. coli in large amounts and to form inclusion bodies. The purified inclusion bodies can then be used in the vaccine composition to induce in vivo production of antibodies that inhibit the activity of native E. corrodens lysine decarboxylase.

Abstract: The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Abstract: The invention relates to sesquiterpene synthases and methods of their production and use. In one embodiment, the invention provides nucleic acids comprising a nucleotide sequence as described herein that encodes for at least one sesquiterpene synthases. In a further embodiment, the invention also provides for sesquiterpene synthases and methods of making and using these enzymes. For example, sesquiterpene synthases of the invention may be used to convert farnesyl-pyrophosphate to various oxygenated and aliphatic sesquiterpenes including valencene, bicyclo-germacrene, cubebol and delta-cadinene.

Abstract: This invention provides a method for combining overlapping DNA molecules comprising: (a) providing first and second DNA fragments, the first having a region homologous to a region in the second; (b) tagging the first DNA fragment with a selectable marker; (c) cloning the first DNA sequence into a retrieval vector to form a DNA-vector complex; (d) linearizing the DNA-vector complex; and (e) inserting the first DNA fragment from the DNA-vector complex into the second DNA fragment using homologous recombination to form a combined DNA molecule; and (f) removing the selectable marker, thereby generating a combined DNA molecule. The invention further provides a vector for retrieving and inserting a selected DNA molecule into a target DNA molecule.

Abstract: The invention provides a method to efficiently express high levels of a recombinant untagged NT-proBNP for use as a calibrator in NT-proBNP immunoassays.

Abstract: The present invention provides a microorganism useful for biologically producing 1,3-propanediol from a fermentable carbon source at higher yield than was previously known. The complexity of the cofactor requirements necessitates the use of a whole cell catalyst for an industrial process that utilizes this reaction sequence to produce 1,3-propanediol. The invention provides a microorganism with disruptions in specified genes and alterations in the expression levels of specified genes that is useful in a higher yielding process to produce 1,3-propanediol.

Abstract: High colicin producing bacteria strains are produced by introducing into a host cell multiple copies of a plasmid containing a colicin gene. A suitable host cell is a bacterium strain, Escherichia coli K-12 and examples of plasmids are pColE1-K53 or pColN-284.

Abstract: The invention relates to a method for preparing closed bacterial ghosts by way of specific interactions between partners of a bioaffinity binding pair, and to the bacterial ghosts which can be obtained in this way. Active compounds can be packed into the closed bacterial ghosts. The closed ghosts can be employed in medicine, in the agricultural sphere and in biotechnology.

Abstract: This invention provides methods and compositions for incorporation of an unnatural amino acid into a peptide using an orthogonal aminoacyl tRNA synthetase/tRNA pair. In particular, an orthogonal pair is provided to incorporate 5-hydroxy-L-tryptophan in a position encoded by an opal mutation.

Abstract: An enteric bacterial strain was engineered to over-produce L-tyrosine using a one-step method. The pheA-tyrA chromosomal region of the bacterial genome was replaced with an engineered chromosomal segment, resulting in inactivation of the pheA coding region and strong expression of the tyrA coding region, resulting in high levels of L-tyrosine production.

Abstract: The present invention provides a Brevundimonas sp. strain SD212-derived peptide having ?-ionone ring-2-hydroxylase activity and a gene encoding the same, to thereby make it possible to produce rare carotenoids in which a hydroxyl group is introduced at the position 2(2?) carbon in their ?-ionone ring in large quantities. The present invention also provides a novel gene encoding an enzyme which introduces a hydroxyl group at the position 3(3?) carbon in the ?-ionone ring of carotenoids, and a novel gene encoding a geranylgeranyl pyrophosphate synthase.

Abstract: Lambda phages that can be used to introduce recombineering functions into host cells are disclosed. Also disclosed are plasmids that can be used to confer recombineering functions to a variety of strains of E. coli and to other bacteria, including Salmonella, Pseudomonas, Cyanobacteria, Spirochaetes. These plasmids and phages can be isolated in vitro and can be used to transform bacterial cells, such as gram negative bacteria.

Abstract: The present invention provides methods of producing an isoprenoid or an isoprenoid precursor in a genetically modified host cell. The methods generally involve modulating the level of hydroxymethylglutaryl-CoA (HMG-CoA) in the cell, such that the level of HMG-CoA is not toxic to the cell and/or does not substantially inhibit cell growth, but is maintained at a level that provides for high-level production of mevalonate, IPP, and other downstream products of an isoprenoid or isoprenoid pathway, e.g., polyprenyl diphosphates and isoprenoid compounds. The present invention further provides genetically modified host cells that are suitable for use in a subject method. The present invention further provides recombinant nucleic acid constructs for use in generating a subject genetically modified host cell, including recombinant nucleic acid constructs comprising nucleotide sequences encoding one or more mevalonate pathway enzymes, and recombinant vectors (e.g., recombinant expression vectors) comprising same.

Abstract: The invention provides methods and compositions for in vivo incorporation of unnatural amino acids. Also provided are compositions including proteins with unnatural amino acids.

Abstract: Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal lysyl-tRNAs, orthogonal lysyl-aminoacyl-tRNA synthetases, and orthogonal pairs of lysyl-tRNAs/synthetases, which incorporate homoglutamines into proteins are provided in response to a four base codon. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with homoglutamines using these orthogonal pairs.

Abstract: The invention relates to a system for stable maintenance of a plasmid, to host cells for use in this system and to methods of using the system to obtain a plasmid useful in medical applications. In particular, the invention provides transformed host cell containing: i) a chromosomal gene which inhibits cell growth; and ii) a plasmid encoding an antisense sequence, wherein the antisense sequence encoded by the plasmid inhibits the action of the chromosomal gene, thereby permitting cell growth and a method for stable maintenance of a plasmid in a host cell in vivo.

Abstract: The present invention provides compositions, including vectors, and methods for the rapid subcloning of nucleic acid sequences in vivo and in vitro. In particular, the invention provides vectors used to contain a gene of interest that comprise a sequence-specific recombinase target site. These vectors are used to rapidly transfer the gene or genes of interest into any vector that contains a sequence-specific recombinase target site located downstream of a regulatory element so that the gene of interest may be regulated.

Abstract: Escherichia coli strains, such as enterotoxigenic E. coli strains, genetically engineered to express from recombinant plasmids one or more colonization factors (CFs) associated with enterotoxigenic Escherichia coli bacteria (ETEC) in an increased amount compared to said CFs expressed by ETEC wild-type reference strains, as well as a method of producing such strains, and vaccine compositions against diarrhea comprising such strains, are described. Further, E. coli strains expressing unnatural combination of at least two different CFs, e.g., CFA/I+CS2, CFA/I+CS6, or CS2+CS4 are disclosed.

Abstract: The present invention is directed to methods and compositions for microbial based production of pravastatin. The compositions of the invention include novel strains of microorganisms that are capable of efficiently hydroxylating compactin (ML-236 B) resulting in production of pravastatin. In particular, the microorganisms of the invention are genetically engineered to express both cytochrome P-450 and the fdxshe or fdxshe-like protein. The invention further relates to the use of such microorganisms in processes designed for production of pravastatin for use in treatment of disease such as hypercholesterolemia and hyperlipidemia.

Abstract: Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Abstract: Novel proteins and their corresponding nucleotide sequences in enteroaggregative Escherichia coli (EAEC) are provided. In particular, Aap and the five gene cluster (aat) of the AA probe region of the pAA plasmid of EAEC 042 have been identified, sequenced, and further characterized. The use of these novel proteins and their corresponding nucleotide sequences for diagnosis, therapy, and prevention of EAEC infections is also provided.

Abstract: L-Glutamic acid, L-proline or L-arginine is produced by culturing a bacterium belonging to the genus Escherichia, which is L-isoleucine auxotrophic and has ability to produce L-glutamic acid, L-proline or L-arginine, in a medium containing L-isoleucine, to produce and accumulate L-glutamic acid, L-proline or L-arginine in a culture, and collecting L-glutamic acid, L-proline or L-arginine from the culture.

Abstract: Novel proteins and their corresponding nucleotide sequences in enteroaggregative Escherichia coli (EAEC) are provided. In particular, Aap and the five gene cluster (aat) of the AA probe region of the pAA plasmid of EAEC 042 have been identified, sequenced, and further characterized. The use of these novel proteins and their corresponding nucleotide sequences for diagnosis, therapy, and prevention of EAEC infections is also provided.

Abstract: The object of the present invention is to provide a novel dehydrogenase having a property which is different from that of known dehydrogenases. The present invention provides a dehydrogenase having the following physicochemical properties: (1) effect: to produce N-alkyl-L-alanine from pyruvic acid and alkylamine or dialkylamine using NADPH and/or NADH as coenzyme; (2) substrate specificity: to show activity to alkylamine or dialkylamine but not to ammonium; (3) optimal pH when using phenylpyruvic acid and methylamine as substrates is around 10; and (4) when treated at 30° C. for 30 minutes, the enzyme is stable at around pH 5 to 10.5.

Abstract: A bacterial high-expression system which is applicable for simultaneous screening of large numbers of recombinant clones from combinatorial antibody libraries is disclosed. The method pertains to screening of single chain antibodies from libraries expressed in the periplasm of E. coli by secretion. By this approach, approximately 104 clones can be screened in a single round. After screening, the clones, which express the recombinant antibodies to the desired antigen, can be directly used for production of large quantities of antibodies from microorganism culture. The system is especially attractive for fast screening of antibody libraries from a hybridoma source. A refolding method for the large-scale production of biologically active scFv-6 his proteins from bacterial inclusion bodies is also disclosed.

Abstract: The invention relates to novel PQQ-dependent soluble glucose dehydrogenases (sPQQGDH) from Acinetobacter and to a process for their preparation by overexpression in suitable microbial expression systems.

Abstract: Methods are provided for curing a host cell of a persistent plasmid of the I-complex super-family. The method proceeds via the direct introduction of a second plasmid containing a gene expressing at least a portion of an I-complex super-family-type Inc RNA. The second plasmid may be cured from the host cell, such as with the use of a temperature sensitive origin of replication.

Abstract: The invention relates to the isolation, sequencing, and recombinant expression of genes encoding either a nitrile hydratase (NHase) or amidase (Am) from Comamonas testosteroni 5-MGAM-4D, where the NHase is useful for catalyzing the hydration of nitriles to the corresponding amides, and the amidase is useful for hydrolysis of amides to the corresponding carboxylic acids. Also provided are transformed host cells containing polynucleotides for expressing the nitrile hydratase or amidase enzymes from Comamonas testosteroni 5-MGAM-4D.

Abstract: Translation systems and other compositions including orthogonal aminoacyl tRNA-synthetases that preferentially charge an orthogonal tRNA with an iodinated or brominated amino acid are provided. Nucleic acids encoding such synthetases are also described, as are methods and kits for producing proteins including heavy atom-containing amino acids, e.g., brominated or iodinated amino acids. Methods of determining the structure of a protein, e.g., a protein into which a heavy atom has been site-specifically incorporated through use of an orthogonal tRNA/aminoacyl tRNA-synthetase pair, are also described.

Abstract: A whole cell catalyst is described comprising a hydantoinase, a racemase and a carbamoylase. Thus this catalyst is able to degrade hydantoins directly into the amino acids. Additionally, a process for the production of this catalysts and for the production of amino acids is claimed.

Abstract: The present invention provides a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family, particularly a bacterium belonging to genus Escherichia or Pantoea, which has been modified to attenuate expression of the yafA gene.

Abstract: An enteric bacterial strain was engineered to over-produce L-tyrosine using a one-step method. The pheA-tyrA chromosomal region of the bacterial genome was replaced with an engineered chromosomal segment, resulting in inactivation of the pheA coding region and strong expression of the tyrA coding region, resulting in high levels of L-tyrosine production.

Abstract: A combined yeast/bacterial two-hybrid system is disclosed.

Abstract: There are disclosed methods and compositions for gene expression and enhancement of protein production and/or accumulation. The invention provides gene-cassettes and methods of introducing the same into host cells for enhanced expression of target genes and production and/or accumulation of encoded proteins or peptides, or the like.

Abstract: The present invention provides a microorganism useful for biologically producing 1,3-propanediol from a fermentable carbon source at higher yield than was previously known. The complexity of the cofactor requirements necessitates the use of a whole cell catalyst for an industrial process that utilizes this reaction sequence to produce 1,3-propanediol. The invention provides a microorganism with disruptions in specified genes and alterations in the expression levels of specified genes that is useful in a higher yielding process to produce 1,3-propanediol.

Abstract: Methods are provided for manipulating nucleic acid to produce gene fusions, to delete or clone a portion of a chromosome, or to insert a sequence into a chromosome. The methods employ sequential transposition processes using two or more pairs of inverted repeat transposase-interacting sequences on a transposable polynucleotide wherein each pair of transposase-interacting sequences interacts with a distinct transposase enzyme.

Abstract: A target substance is produced by culturing a bacterium which has the ability to produce the target substance in a medium to cause accumulation of said target substance in the medium and collecting the target substance from the medium, wherein the bacterium is modified so that a system for uptake of a byproduct of the target substance or a substrate for a biosynthesis system of the target substance into the bacterial cell.

Abstract: The present invention relates to polypeptides having glucoamylase activity and isolated polynucleotides encoding said polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides. The invention also relates to the composition comprising a glucoamylase of the invention as well as the use such compositions for starch conversion processes, brewing, including processes for producing fermentation products or syrups.

Abstract: The invention provides methods and materials related to the production of organic products such as glucuronic acid, ascorbic acid, and glucaric acid. Specifically, the invention provides cells, methods for culturing cells, isolated nucleic acid molecules, and methods and materials for producing various organic products such as glucuronic acid, ascorbic acid, and glucaric acid.

Abstract: Methods for the production of PHCA are disclosed. The methods rely on the tight gene expression of genetic constructs encoding polypeptides having tyrosine ammonia lyase activity. Promoters of choice are arabinose inducible.

Abstract: The present invention relates to a method and a kit for assessing mutability of a DNA sequence of interest. The method involves using a mutation hotspot sequence as a standard to determine whether the DNA sequence of interest is more or less mutable than the hotspot sequence. The mutation events are detected using a bacterial system in which the DNA sequence of interest and the mutation hotspot sequence are each linked in-frame to a reporter gene such as a killer gene or a color gene so that any nonsense or out-of-frame frame shit mutation in the DNA sequence of interest or the mutation hotspot sequence can be reflected by a loss of the function of the reporter gene product. The kit of present invention contains one or more of the various vectors that are useful for practicing the method disclosed herein.

Abstract: 4-vinylguaiacol is produced using recombinant E. coli containing a decarboxylase gene from Bacillus pumilis in an aqueous fermentation broth and in an immobilized whole cell system. The 4-vinylguaiacol is extracted and recovered from an organic hydrocarbon solvent, preferably n-octane, whereby the product can readily be separated.

Abstract: The invention relates to sesquiterpene synthases and methods of their production and use. In one embodiment, the invention provides nucleic acids comprising a nucleotide sequence as described herein that encodes for at least one sesquiterpene synthases. In a further embodiment, the invention also provides for sesquiterpene synthases and methods of making and using these enzymes. For example, sesquiterpene synthases of the invention may be used to convert farnesyl-pyrophosphate to various oxygenated and aliphatic sesquiterpenes including valencene, bicyclo-germacrene, cubebol and delta-cadinene.

Abstract: This invention relates to a method for the production of D-(?)-3-hydroxybutyric acid, comprising the step of culturing a recombinant strain containing genes phbA, phbB, ptb and buk by fermentation. Preferably, the recombinant strain is a strain of E. coli. The method of the invention is simple, avoiding the technique of degrading polymer to produce D-(?)-3-hydroxybutyric acid. The present method also provides improved efficiency, lowers the complicated requirement for facilities as used in traditional chemical synthesis, simplifies the complicated technique flow, and omits the complicated chiral separation step. Therefore, the present method greatly reduces the costs associated with D-(?)-3-hydroxybutyric acid production. Also, with this invention, the problems such as environmental pollution of chemical synthesis and chiral separation are overcome.

Abstract: Methods of increasing yields of succinate using aerobic culture methods and a multi-mutant E. coli strain are provided. Also provided is a mutant strain of E. coli that produces high amounts of succinic acid.

Abstract: The invention relates to a process for the preparation of L-amino acids, especially L-lysine, L-valine, L-homoserine and L-threonine, by fermenting a microorganism of the genus Escherichia which has a mutation or deletion in the gene encoding aspartate ammonium lyase (aspA).

Abstract: Methods of increasing yields of succinate using aerobic culture methods and a multi-mutant E. coli strain are provided. Also provided is a mutant strain of E. coli that produces high amounts of succinic acid.

Abstract: The complete nucleotide sequence of the genome of porcine adenovirus type 3 (PAV-3) is provided. Methods for construction of infectious PAV genomes by homologous recombination in procaryotic cells are provided. Recombinant PAV viruses are obtained by transfection of mammalian cells with recombinant PAV genomes. The PAV-3 genome can be used as a vector for the expression of heterologous nucleotide sequences, for example, for the preparation and administration of subunit vaccines to swine or other mammals. In addition, PAV-3 vectors can be used for gene therapy and expression of heterologous polypeptides. PAV-3 genome sequences can also be used for diagnostic purposes, to detect the presence of PAV-3 DNA in a subject or biological sample.