Publication Date:
2008-01-26
Description:
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.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Gibson, Daniel G -- Benders, Gwynedd A -- Andrews-Pfannkoch, Cynthia -- Denisova, Evgeniya A -- Baden-Tillson, Holly -- Zaveri, Jayshree -- Stockwell, Timothy B -- Brownley, Anushka -- Thomas, David W -- Algire, Mikkel A -- Merryman, Chuck -- Young, Lei -- Noskov, Vladimir N -- Glass, John I -- Venter, J Craig -- Hutchison, Clyde A 3rd -- Smith, Hamilton O -- New York, N.Y. -- Science. 2008 Feb 29;319(5867):1215-20. doi: 10.1126/science.1151721. Epub 2008 Jan 24.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉J. Craig Venter Institute, Rockville, MD 20850, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/18218864" target="_blank"〉PubMed〈/a〉
Keywords:
Base Sequence
;
Chromosomes, Artificial, Bacterial
;
Chromosomes, Artificial, Yeast
;
*Cloning, Molecular
;
DNA, Bacterial/*chemical synthesis
;
DNA, Recombinant
;
Escherichia coli/genetics
;
Genetic Vectors
;
*Genome, Bacterial
;
Genomics/*methods
;
Mycoplasma genitalium/*genetics
;
Oligodeoxyribonucleotides/chemical synthesis
;
Plasmids
;
Recombination, Genetic
;
Saccharomyces cerevisiae/genetics
;
Sequence Analysis, DNA
;
Transformation, Genetic
Print ISSN:
0036-8075
Electronic ISSN:
1095-9203
Topics:
Biology
,
Chemistry and Pharmacology
,
Computer Science
,
Medicine
,
Natural Sciences in General
,
Physics
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