synthetic Mycoplasma genitalium JCVI-1.0. The genome, which was designed based on Genbank Accession Number L43967 (Mycoplasma genitalium G37), was assembled from chemically synthesized oligonucleotides.
More... synthetic Mycoplasma genitalium JCVI-1.0. The genome, which was designed based on Genbank Accession Number L43967 (Mycoplasma genitalium G37), was assembled from chemically synthesized oligonucleotides. The sequenced genome was isolated from a yeast clone.
This synthetic genome contains all of the genes of wild type M. genitalium G37 except MG408, which is disrupted to block pathogenicity. The genome also contains added watermark sequences located at intergenic sites known to tolerate transposon insertions, for identification of the genome as synthetic and also antibiotic resistance markers to allow for selection. Overlapping cassettes of 5-7 kb, assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of ~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 (TAR) cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. This synthetic M. genitalium genome can be isolated for future work. Less...