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Published Online January 24, 2008
Science DOI: 10.1126/science.1151721

Research Articles

Submitted on October 15, 2007
Accepted on January 11, 2008

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

Daniel G. Gibson 1, Gwynedd A. Benders 1, Cynthia Andrews-Pfannkoch 1, Evgeniya A. Denisova 1, Holly Baden-Tillson 1, Jayshree Zaveri 1, Timothy B. Stockwell 1, Anushka Brownley 1, David W. Thomas 1, Mikkel A. Algire 1, Chuck Merryman 1, Lei Young 1, Vladimir N. Noskov 1, John I. Glass 1, J. Craig Venter 1, Clyde A. Hutchison III1, Hamilton O. Smith 1*

1 The J. Craig Venter Institute, Rockville, MD 20850, USA.

* To whom correspondence should be addressed.
Hamilton O. Smith , E-mail: hsmith{at}jcvi.org

We have synthesized a 582,970 bp 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 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 (BACs) 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 (TAR) 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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