Synthetic Genomics, Inc.

Codex Dna, Inc.

Telesis Bio Inc.

Sgi-dna, Inc.

Keene Building Products Co., Inc.

Air Products and Chemicals, Inc.

Daniel Glenn Gibson

Hamilton O Smith

J Craig Venter

Clyde A Hutchison

Lei Young

John E Gill

Vladimir N Noskov

Ray-Yuan Chuang

Nicky Caiazza

John Irvin Glass

Gwynedd A Benders

Lixia Fu

Krishna Kannan

Toby Howard Richardson

Laurence Warden

Matthew T Weinstock

Bogumil J Karas

Carole Lartigue

Sanjay Vashee

Charles E Merryman

Daniel Strimling

Tei Newman-Lehman

Kim S Wise

John I Glass

Mikkel A Algire

Chuck Merryman

Lin Ding

James R Keene

Ranajit Ghosh

Jun Urano

John Lewis Green

Kevin John Giardiniere

Toby H Richardson

Craig J Venter

Clyde A Iii Hutchison

Clyde A Hutchison Iii

Zurecki Zbigniew

Zhiqing Qi

Nicky C Caiazza

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.

Methods for in vitro joining and combinatorial assembly of nucleic acid molecules

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.

Generation of synthetic genomes

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. 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.

Demand synthesis of polynucleotide sequences

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.

Methods for assembling nucleic acids

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. Also provided are novel nucleic acid sequences encoding mismatch endonucleases, polypeptides encoded thereby, as well as nucleic acid constructs, transgenic cells, and various compositions thereof.

Materials and methods for the synthesis of error-minimized nucleic acid molecules

The invention provides engineeredsp. 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.

Methods of cloning nucleic acids or producing proteins in a low endotoxin organism

The present invention provides a system for receiving biological sequence information and activating the synthesis of a biological entity. The system has a receiving unit for receiving a signal encoding biological sequence information transmitted from a transmitting unit. The transmitting unit can be present at a remote location from the receiving unit. The system also has an assembly unit connected to the receiving unit, and the assembly unit assembles the biological entity according to the biological sequence information. Thus, according to the present invention biological sequence information can be digitally transmitted to a remote location and the information converted into a biological entity, for example a protein useful as a vaccine, immediately upon being received by the receiving unit and without further human intervention after preparing the system for receipt of the information. The invention is useful, for example, for rapidly responding to viral and other biological threats that are specific to a particular locale.

Digital to biological converter

The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5' exonuclease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.

In vitro recombination method

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&#x2032; 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&#x2032; flanking sequence and a universal 5&#x2032; flanking sequence, respectively. The methods involve performing a first amplification reaction on the plurality of pairs of oligonucleotides; removing the 3&#x2032; and 5&#x2032; primer binding sequences from the plurality of pairs of oligonucleotides; and subjecting the plurality of pairs of oligonucleotides to an assembly reaction to thereby assemble the dsDNA molecule having the desired sequence.

Methods for assembling DNA molecules

Recombinant Vibrio natriegens organisms having a modified outer membrane

Growing community of inventors

Crofton, MD, United States of America

Daniel Glenn Gibson