One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome.
We previously reported assembly and cloning of the synthetic Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharomyces cerevisiae by recombination of six overlapping DNA fragments to produce a 592-kb circle. Here we extend this approach by demonstrating assembly of the synthetic genome from 25 overlapping fragments in a single step. The use of yeast recombination greatly simplifies the assembly of large DNA molecules from both synthetic and natural fragments.
Gibson
Daniel G
DG
The J. Craig Venter Institute, Synthetic Biology Group, Rockville, MD 20850, USA. dgibson@jcvi.org
Benders
Gwynedd A
GA
Axelrod
Kevin C
KC
Zaveri
Jayshree
J
Algire
Mikkel A
MA
Moodie
Monzia
M
Montague
Michael G
MG
Venter
J Craig
JC
Smith
Hamilton O
HO
Hutchison
Clyde A
CA
D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000096:biosynthesis*; D016680:Genome, Bacterial / Q000235:genetics*; D045704:Mycoplasma genitalium / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D015003:Yeasts / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D004247:DNA