Chemical synthesis of the Mycoplasma genitalium genome
Overview
We have developed methods for assembly of the complete synthetic 582,970 bp Mycoplasma genitalium JCVI 1.0 genome. This synthetic genome contains all 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 its selection. Overlapping “cassettes” of 5-7 kb, assembled from chemically synthesized oligonucleotides, have been 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 has been assembled from quarter molecules by transformation associated recombination cloning in the yeast Saccharomyces cerevisiae. The complete genome has been isolated and is being sequenced. Methods for isolation of microgram quantities of the synthetic M. genitalium genome are under development. Experiments are underway to transplant this synthetic genome into a receptive mycoplasma cytoplasm such that a new cell operating with the synthetic chromosome can be grown in the laboratory.
Funding