Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome

American Association for the Advancement of Science (AAAS) - Tập 319 Số 5867 - Trang 1215-1220 - 2008
Daniel G. Gibson1, Gwynedd A. Benders1, Cynthia Andrews‐Pfannkoch1, Evgeniya A. Denisova1, Holly Baden-Tillson1, Jayshree Zaveri1, Timothy B. Stockwell1, Anushka Brownley1, David W. Thomas1, Mikkel A. Algire1, Chuck Merryman1, Lei Young1, Vladimir N. Noskov1, John I. Glass1, J. Craig Venter1, Clyde A. Hutchison1, Hamilton O. Smith1
1The J. Craig Venter Institute Rockville MD 20850 USA

Tóm tắt

We have synthesized a 582,970–base pair Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted “watermarks” at intergenic sites known to tolerate transposon insertions. Overlapping “cassettes” of 5 to 7 kilobases (kb), assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb (“1/8 genome”), and 144 kb (“1/4 genome”), which were all cloned as bacterial artificial chromosomes in Escherichia coli . Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae , then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments.

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We thank J. Mulligan for his interest in our work and expediting cassette synthesis by Blue Heron Technologies S. Vashee and R.-Y. Chuang for many helpful discussions about the manuscript and J. Johnson and T. Davidsen for assistance with GenBank submissions. Additionally we thank the Larionov laboratory at NIH for their gifts of yeast strains and TAR cloning expertise. The bulk of the work was supported by Synthetic Genomics Inc. J.C.V. is Chief Executive Officer and Co-Chief Scientific Officer of Synthetic Genomics Inc. a privately held entity that develops genomic-driven strategies to address global energy and environmental challenges. H.O.S. is Co-Chief Scientific Officer and on the Board of Directors of Synthetic Genomics Inc. C.A.H. is Chairman of the Synthetic Genomics Inc. Scientific Advisory Board. All three of these authors hold Synthetic Genomics Inc. stock.

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American Association for the Advancement of Science (AAAS)

Tập 319 Số 5867

1215-1220

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