title: Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome.
abstract: 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.
authors.affiliation: J. Craig Venter Institute, Rockville, MD 20850, USA.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Gibson
authors.forename: Gwynedd A
authors.initials: GA
authors.lastname: Benders
authors.forename: Cynthia
authors.initials: C
authors.lastname: Andrews-Pfannkoch
authors.forename: Evgeniya A
authors.initials: EA
authors.lastname: Denisova
authors.forename: Holly
authors.initials: H
authors.lastname: Baden-Tillson
authors.forename: Jayshree
authors.initials: J
authors.lastname: Zaveri
authors.forename: Timothy B
authors.initials: TB
authors.lastname: Stockwell
authors.forename: Anushka
authors.initials: A
authors.lastname: Brownley
authors.forename: David W
authors.initials: DW
authors.lastname: Thomas
authors.forename: Mikkel A
authors.initials: MA
authors.lastname: Algire
authors.forename: Chuck
authors.initials: C
authors.lastname: Merryman
authors.forename: Lei
authors.initials: L
authors.lastname: Young
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Noskov
authors.forename: John I
authors.initials: JI
authors.lastname: Glass
authors.forename: J Craig
authors.initials: JC
authors.lastname: Venter
authors.forename: Clyde A
authors.initials: CA
authors.lastname: Hutchison
authors.forename: Hamilton O
authors.initials: HO
authors.lastname: Smith
mesh_terms: D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000138:chemical synthesis*; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods*; D045704:Mycoplasma genitalium / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010957:Plasmids; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic
chemical_list: D004269:DNA, Bacterial; D004274:DNA, Recombinant; D009838:Oligodeoxyribonucleotides