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

@article{citeulike:2291542, abstract = {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. 10.1126/science.1151721}, author = {Gibson, Daniel G. and Benders, Gwynedd A. and Andrews-Pfannkoch, Cynthia and Denisova, Evgeniya A. and Baden-Tillson, Holly and Zaveri, Jayshree and Stockwell, Timothy B. and Brownley, Anushka and Thomas, David W. and Algire, Mikkel A. and Merryman, Chuck and Young, Lei and Noskov, Vladimir N. and Glass, John I. and Venter, Craig J. and Hutchison, Clyde A. and Smith, Hamilton O. }, citeulike-article-id = {2291542}, doi = {10.1126/science.1151721}, journal = {Science}, keywords = {assembly, genome, sequence}, month = {January}, pages = {1151721+}, posted-at = {2008-04-12 13:30:43}, priority = {2}, title = {Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome}, url = {http://dx.doi.org/10.1126/science.1151721}, year = {2008} }

TY - JOUR ID - citeulike:2291542 L3 - citeulike-article-id:2291542 TI - Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome JF - Science SP - 1151721 N2 - 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. 10.1126/science.1151721 KW - assembly KW - genome KW - sequence AU - Gibson, Daniel AU - Benders, Gwynedd AU - Andrews-Pfannkoch, Cynthia AU - Denisova, Evgeniya AU - Baden-Tillson, Holly AU - Zaveri, Jayshree AU - Stockwell, Timothy AU - Brownley, Anushka AU - Thomas, David AU - Algire, Mikkel AU - Merryman, Chuck AU - Young, Lei AU - Noskov, Vladimir AU - Glass, John AU - Venter, Craig AU - Hutchison, Clyde AU - Smith, Hamilton PY - 2008/01/24/ UR - http://dx.doi.org/10.1126/science.1151721 ER -