Abstract: The present invention provides materials and methods useful for error correction of nucleic acid molecules. In one embodiment of the invention, a first plurality of double-stranded nucleic acid molecules having a nucleotide mismatch are fragmented by exposure to a molecule having unidirectional mismatch endonuclease activity. The nucleic acid molecules are cut at the mismatch site or near the mismatch site, leaving a double-stranded nucleic acid molecule having a mismatch at the end or near end of the molecule. The nucleic acid molecule is then exposed to a molecule having unidirectional exonuclease activity to remove the mismatched nucleotide. The missing nucleotides can then be filled in by the action of, e.g., a molecule having DNA polymerase activity. The result is double-stranded nucleic acid molecules with a decreased frequency of nucleotide mismatches.

Abstract: The invention provides engineered Vibrio sp. organisms that comprise a genetic modification to either or both of the lpxL and/or lpxM genes. The organisms score substantially lower in an in vitro endotoxin assay versus the unmodified or wild type organism. The organisms preserve substantially the growth rate of the corresponding unmodified organisms. The organisms can also have an exogenous nucleic acid cloned in the organism, or an exogenous nucleic acid encoding a protein, polypeptide, or peptide expressed by the organism, and optionally secreted from the organism.

Abstract: The invention provides methods for synthesizing a product DNA molecule of any possible DNA sequence from a universal library of overlapping oligonucleotides. The method involves combining a plurality of the overlapping oligonucleotides in a reaction pool, where the sequences of the plurality of oligonucleotides comprise at least a sub-sequence of the product DNA molecule. The method also involves annealing the plurality of oligonucleotides, performing a ligation step, and performing an amplification step to thereby synthesize a sub-sequence of the product DNA molecule. The invention can be used to synthesize a DNA molecule of any possible sequence from the universal library, which can be accomplished through a hierarchal assembly scheme. In one embodiment the universal library comprises fewer than 10,000 pre-manufactured oligonucleotides that can be synthesized into the any possible DNA sequence. In any embodiment the product DNA molecule has an error rate of less than 1 error per 2,000 nucleotides.

Abstract: The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.

Abstract: The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.

Abstract: The invention provides methods of synthesizing a product DNA molecule having a desired and/or defined sequence. The methods involve annealing at least one long oligonucleotide and at least one short oligonucleotide to at least one anchor strand having a sequence at least partially complementary to the at least one long and at least one short oligonucleotide. After annealing, at least one long oligonucleotide bound to an anchor strand abuts at least one short oligonucleotide bound to the same anchor strand. The anchor strand has one or more non-standard nucleotides, and optionally one or more degenerate nucleotides. The method involves ligating the abutting at least one long oligonucleotide and at least one short oligonucleotide to form a dsDNA molecule. The invention also provides methods of synthesizing DNA molecules by assembling oligonucleotide members of a library that contains less than 20,000 members that can be assembled into all possible DNA sequences.

Abstract: The invention provides compositions and methods for assembling a DNA molecule having a desired sequence. The methods involve contacting a DNA polymerase, dNTPs, and a plurality of pairs of oligonucleotides. The oligonucleotides of a pair have a portion of the desired sequence, and an internal sequence that overlaps and is complementary to an internal sequence of the other oligonucleotide of the pair, and, when arranged in order, they have at least a portion of the desired sequence. The oligonucleotides also have a 3? or a 5? primer binding sequence having a binding site for a primer. The oligonucleotides that correspond to the end oligonucleotides of the desired sequence also have a universal 3? flanking sequence and a universal 5? flanking sequence, respectively.

Abstract: The invention provides engineered Vibrio sp. organisms that comprise a genetic modification to either or both of the lpxL and/or lpxM genes. The organisms score substantially lower in an in vitro endotoxin assay versus the unmodified or wild type organism. The organisms preserve substantially the growth rate of the corresponding unmodified organisms. The organisms can also have an exogenous nucleic acid cloned in the organism, or an exogenous nucleic acid encoding a protein, polypeptide, or peptide expressed by the organism, and optionally secreted from the organism.

Abstract: The invention provides methods of assembling a DNA molecule having a desired sequence. The methods involve contacting a DNA ligase with a plurality of short oligonucleotides to be assembled and performing the ligase chain reaction to thereby generate a set of polynucleotides. Oligonucleotides in the plurality overlap with and are complementary to a sequence of at least one other oligonucleotide in the plurality, and at least 50% of the oligonucleotides in the plurality are 6-30 nucleotides in length. The set of polynucleotides produced are contacted with a DNA polymerase and dNTPs in a mixture to join the set of polynucleotides and thereby create a DNA molecule having a desired sequence by polymerase chain assembly. The method allows for production of oligonucleotides of any length having very high sequence fidelity to a desired sequence.

Abstract: Methods for generating synthetic genomes, for example synthetic genomes having desired properties or viable genomes of reduced size, are disclosed. Also disclosed are synthetic genomes produced by the methods disclosed herein and synthetic cells containing the synthetic genomes disclosed herein.

Abstract: Methods for generating synthetic genomes, for example synthetic genomes having desired properties or viable genomes of reduced size, are disclosed. Also disclosed are synthetic genomes produced by the methods disclosed herein and synthetic cells containing the synthetic genomes disclosed herein.

Abstract: The invention provides compositions and methods for assembling a DNA molecule having a desired sequence. The methods involve contacting a DNA polymerase, dNTPs, and a plurality of pairs of oligonucleotides. The oligonucleotides of a pair have a portion of the desired sequence, and an internal sequence that overlaps and is complementary to an internal sequence of the other oligonucleotide of the pair, and, when arranged in order, they have at least a portion of the desired sequence. The oligonucleotides also have a 3? or a 5? primer binding sequence having a binding site for a primer. The oligonucleotides that correspond to the end oligonucleotides of the desired sequence also have a universal 3? flanking sequence and a universal 5? flanking sequence, respectively.

Abstract: The invention provides engineered Vibrio sp. organisms that comprise a genetic modification to either or both of the lpxL and/or lpxM genes. The organisms score substantially lower in an in vitro endotoxin assay versus the unmodified or wild type organism. The organisms preserve substantially the growth rate of the corresponding unmodified organisms. The organisms can also have an exogenous nucleic acid cloned in the organism, or an exogenous nucleic acid encoding a protein, polypeptide, or peptide expressed by the organism, and optionally secreted from the organism.

Abstract: The present invention provides materials and methods useful for error correction of nucleic acid molecules. In one embodiment of the invention, a first plurality of double-stranded nucleic acid molecules having a nucleotide mismatch are fragmented by exposure to a molecule having unidirectional mismatch endonuclease activity. The nucleic acid molecules are cut at the mismatch site or near the mismatch site, leaving a double-stranded nucleic acid molecule having a mismatch at the end or near end of the molecule. The nucleic acid molecule is then exposed to a molecule having unidirectional exonuclease activity to remove the mismatched nucleotide. The missing nucleotides can then be filled in by the action of, e.g., a molecule having DNA polymerase activity. The result is double-stranded nucleic acid molecules with a decreased frequency of nucleotide mismatches.

Abstract: The present invention provides materials and methods useful for error correction of nucleic acid molecules. In one embodiment of the invention, a first plurality of double-stranded nucleic acid molecules having a nucleotide mismatch are fragmented by exposure to a molecule having unidirectional mismatch endonuclease activity. The nucleic acid molecules are cut at the mismatch site or near the mismatch site, leaving a double-stranded nucleic acid molecule having a mismatch at the end or near end of the molecule. The nucleic acid molecule is then exposed to a molecule having unidirectional exonuclease activity to remove the mismatched nucleotide. The missing nucleotides can then be filled in by the action of, e.g., a molecule having DNA polymerase activity. The result is double-stranded nucleic acid molecules with a decreased frequency of nucleotide mismatches.

Abstract: The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.

Abstract: The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.

Abstract: The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.

Abstract: The present invention discloses methods for assembling a nucleic acid molecule from a set of overlapping oligonucleotides. The method involves contacting a set of overlapping oligonucleotides with a DNA polymerase, a mixture of dNTPs, and a crowding agent to form an assembly mixture. In one embodiment the crowding agent is polyethylene glycol (PEG). The presence of the crowding agent facilitates the nucleic acid assembly process of the invention. The assembly mixture is then subjected to multiple cycles, each cycle comprising an annealing phase, an extension phase, and a denaturation phase, and the desired nucleic acid molecule is thereby assembled. In some embodiments one or more of the phases are time varied.

Abstract: The invention provides engineered Vibrio sp. organisms that comprise a genetic modification to either or both of the lpxL and/or lpxM genes. The organisms score substantially lower in an in vitro endotoxin assay versus the unmodified or wild type organism. The organisms preserve substantially the growth rate of the corresponding unmodified organisms. The organisms can also have an exogenous nucleic acid cloned in the organism, or an exogenous nucleic acid encoding a protein, polypeptide, or peptide expressed by the organism, and optionally secreted from the organism.