Synthetic Genomics, Inc.

Other

Codex Dna, Inc.

Human Genome Sciences, Inc.

Applera Corporation

Sgi-dna, Inc.

The Johns Hopkins University

Hamilton O Smith

J Craig Venter

Daniel Glenn Gibson

Clyde A Hutchison

Lei Young

Owen White

Mark D Adams

Robert D Fleischmann

John Irvin Glass

Ray-Yuan Chuang

Vladimir N Noskov

Carole Lartigue

Bogumil J Karas

Clyde A Hutchison, Iii

Gwynedd A Benders

Charles E Merryman

Krishna Kannan

Sanjay Vashee

Chuck Merryman

Mikkel A Algire

Lin Ding

Kim S Wise

Robert Holt

John I Glass

Cynthia Pfannkoch

Carl R Woese

Carol J Bult

Claire M Fraser

Jeannine D Gocayne

Qing Xu

Nina Alperovich

Nacyra Assad-Garcia

Hutchison Clyde A, Iii

Yo Suzuki

Michael G Montague

Clyde A Iii Hutchison

Craig Venter

Craig J Venter

Charles Merryman

Clyde A Iii Hutchinson

Clyde A Hutchison Iii

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 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

Methods and apparatus are disclosed herein for encoding human readable text conveying a non-genetic message into nucleic acid sequences with a substantially reduced probability of biological impact and decoding such text from nucleic acid sequences. In one embodiment, each symbol of a symbol set of human readable symbols uniquely maps to a respective codon identifier. Mapping may ensure that each symbol will not map to a codon identifier that generates an amino acid residue which has a single-letter abbreviation that is the equivalent to the respective symbol. Synthetic nucleic acid sequences comprising such human readable text, and recombinant or synthetic cells comprising such sequences are provided, as well as methods of identifying cells, organisms, or samples containing such sequences.

Encoding text into nucleic acid sequences

Methods are provided for constructing a synthetic genome, comprising generating and assembling nucleic acid cassettes comprising portions of the genome, wherein at least one of the nucleic acid cassettes is constructed from nucleic acid components that have been chemically synthesized, or from copies of the chemically synthesized nucleic acid components. In one embodiment, the entire synthetic genome is constructed from nucleic acid components that have been chemically synthesized, or from copies of the chemically synthesized nucleic acid components. Rational methods may be used to design the synthetic genome (e.g., to establish a minimal genome and/or to optimize the function of genes within a genome, such as by mutating or rearranging the order of the genes). Synthetic genomes of the invention may be introduced into vesicles (e.g., bacterial cells from which part or all of the resident genome has been removed, or synthetic vesicles) to generate synthetic cells. Synthetic genomes or synthetic cells may be used for a variety of purposes, including the generation of synthetic fuels, such as hydrogen or ethanol.

Method of nucleic acid cassette assembly

A method to assemble any desired nucleic acid molecule by combining cassettes in vitro to form assemblies which are further combined in vivo, or by assembling large numbers of DNA fragments by recombination in a yeast culture to obtain desired DNA molecules of substantial size is described.

Assembly of large nucleic acids

A method is provided for introducing a genome into a cell or cell-like system. The introduced genome may occur in nature, be manmade with or without automation, or may be a hybrid of naturally occurring and manmade materials. The genome is obtained outside of a cell with minimal damage. Materials such as a proteins, RNAs, polycations, nucleoid condensation proteins, or gene translation systems may accompany the genome. The genome is installed into a naturally occurring cell or into a manmade cell-like system. A cell-like system or synthetic cell resulting from the practice of the provided method may be designed and used to yield gene-expression products, such as desired proteins. By enabling the synthesis of cells or cell-like systems comprising a wide variety of genomes, accompanying materials and membrane types, the provided method makes possible a broader field of experimentation and bioengineering than has been available using prior art methods.

Installation of genomes or partial genomes into cells or cell-like systems

Compositions and methods are disclosed herein for cloning a donor genome in a heterologous host cell. In one embodiment, the donor genome can be further modified within a host cell. Modified or unmodified genomes can be further isolated from the host cell and transferred to a recipient cell. Methods disclosed herein can be used to alter donor genomes from intractable donor cells in more tractable host cells.

Methods for cloning and manipulating genomes

Compositions and methods are disclosed herein for cloning a synthetic or a semi-synthetic donor genome in a heterologous host cell. In one embodiment, the donor genome can be further modified within a host cell. Modified or unmodified genomes can be further isolated from the host cell and transferred to a recipient cell. Methods disclosed herein can be used to alter donor genomes from intractable donor cells in more tractable host cells.

Growing community of inventors

San Diego, CA, United States of America

Hamilton O Smith