Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome.

摘要: We have synthesized a 582,970 bp Mycoplasma genitalium genome. This synthetic genome, named M. JCVI-1.0, contains all the genes of wild-type G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and allow for selection. To identify genome as synthetic, we inserted “watermarks” at intergenic sites known tolerate transposon insertions. Overlapping “cassettes” 5 7 kb, assembled from chemically oligonucleotides, were joined in vitro recombination produce intermediate assemblies approximately 24 72 kb (“1/8 genome”), 144 (“1/4 cloned bacterial artificial chromosomes (BACs) Escherichia coli. Most these clones sequenced, four 1/4 genomes with correct sequence identified. The complete transformationassociated (TAR) cloning yeast Saccharomyces cerevisiae, then isolated sequenced. A clone methods described here will be generally useful constructing large DNA molecules pieces also combinations natural segments. is bacterium smallest any independently replicating cell that has been grown pure