Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome
Abstract
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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Science
Volume 319 | Issue 5867
29 February 2008
29 February 2008
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American Association for the Advancement of Science.
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Received: 15 October 2007
Accepted: 11 January 2008
Published in print: 29 February 2008
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