Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome.
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.
Gibson
Daniel G
DG
J. Craig Venter Institute, Rockville, MD 20850, USA.
Benders
Gwynedd A
GA
Andrews-Pfannkoch
Cynthia
C
Denisova
Evgeniya A
EA
Baden-Tillson
Holly
H
Zaveri
Jayshree
J
Stockwell
Timothy B
TB
Brownley
Anushka
A
Thomas
David W
DW
Algire
Mikkel A
MA
Merryman
Chuck
C
Young
Lei
L
Noskov
Vladimir N
VN
Glass
John I
JI
Venter
J Craig
JC
Hutchison
Clyde A
CA
Smith
Hamilton O
HO
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
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D009838:Oligodeoxyribonucleotides