Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.
Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome
Daniel G. Gibson,Gwynedd A. Benders,Cynthia Andrews-Pfannkoch,Evgeniya A. Denisova,Holly Baden-Tillson,Jayshree Zaveri,Timothy B. Stockwell,Anushka Brownley,David W. Thomas,Mikkel A. Algire,Chuck Merryman,Lei Young,Vladimir N. Noskov,John I. Glass,J. Craig Venter,Clyde A. Hutchison, III,Hamilton O. Smith*
We have synthesized a 582,970–base pair Mycoplasma genitaliumgenome. This synthetic genome, named M. genitalium JCVI-1.0,contains all the genes of wild-type M. genitalium G37 exceptMG408, which was disrupted by an antibiotic marker to blockpathogenicity and to allow for selection. To identify the genomeas synthetic, we inserted "watermarks" at intergenic sites knownto tolerate transposon insertions. Overlapping "cassettes" of5 to 7 kilobases (kb), assembled from chemically synthesizedoligonucleotides, were joined by in vitro recombination to produceintermediate assemblies of approximately 24 kb, 72 kb ("1/8genome"), and 144 kb ("1/4 genome"), which were all cloned asbacterial artificial chromosomes in Escherichia coli. Most ofthese intermediate clones were sequenced, and clones of allfour 1/4 genomes with the correct sequence were identified.The complete synthetic genome was assembled by transformation-associatedrecombination cloning in the yeast Saccharomyces cerevisiae,then isolated and sequenced. A clone with the correct sequencewas identified. The methods described here will be generallyuseful for constructing large DNA molecules from chemicallysynthesized pieces and also from combinations of natural andsynthetic DNA segments.
The J. Craig Venter Institute, Rockville, MD 20850, USA.
* To whom correspondence should be addressed. E-mail: hsmith{at}jcvi.org
TmPrime: fast, flexible oligonucleotide design software for gene synthesis.
M. Bode, S. Khor, H. Ye, M.-H. Li, and J. Y. Ying (2009)
Nucleic Acids Res.
37, W214-W221
|Abstract »|Full Text »|PDF »
Creation of a type IIS restriction endonuclease with a long recognition sequence.
S. M. Lippow, P. M. Aha, M. H. Parker, W. J. Blake, B. M. Baynes, and D. Lipovsek (2009)
Nucleic Acids Res.
37, 3061-3073
|Abstract »|Full Text »|PDF »
Experimental analysis of gene assembly with TopDown one-step real-time gene synthesis.
H. Ye, M. C. Huang, M.-H. Li, and J. Y. Ying (2009)
Nucleic Acids Res.
37, e51
|Abstract »|Full Text »|PDF »
DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways.
Expanding metabolism for biosynthesis of nonnatural alcohols.
K. Zhang, M. R. Sawaya, D. S. Eisenberg, and J. C. Liao (2008)
PNAS
105, 20653-20658
|Abstract »|Full Text »|PDF »
One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome.
D. G. Gibson, G. A. Benders, K. C. Axelrod, J. Zaveri, M. A. Algire, M. Moodie, M. G. Montague, J. C. Venter, H. O. Smith, and C. A. Hutchison III (2008)
PNAS
105, 20404-20409
|Abstract »|Full Text »|PDF »
Synthetic recombinant bat SARS-like coronavirus is infectious in cultured cells and in mice.
M. M. Becker, R. L. Graham, E. F. Donaldson, B. Rockx, A. C. Sims, T. Sheahan, R. J. Pickles, D. Corti, R. E. Johnston, R. S. Baric, et al. (2008)
PNAS
105, 19944-19949
|Abstract »|Full Text »|PDF »
Transcription of foreign DNA in Escherichia coli.
R. L. Warren, J. D. Freeman, R. C. Levesque, D. E. Smailus, S. Flibotte, and R. A. Holt (2008)
Genome Res.
18, 1798-1805
|Abstract »|Full Text »|PDF »
