Synthetic Mycoplasma mycoides JCVI-syn1.0. The genome, which was designed based on Genbank Accession Number CP001668 Mycoplasma mycoides subspecies capri strain GM12), was assembled from chemically synthesized oligonucleotides. More...
Synthetic Mycoplasma mycoides JCVI-syn1.0. The genome, which was designed based on Genbank Accession Number CP001668 Mycoplasma mycoides subspecies capri strain GM12), was assembled from chemically synthesized oligonucleotides. The sequenced genome was isolated from a yeast clone. The sequenced clone is sMmYCp235-1.
This synthetic genome contains all but 14 of the genes of wild type Mycoplasma mycoides subspecies capri strain GM12. Among the genes deleted from the genome are components of the ABC glycerol transporter operon gtsABCD. These genes are involved in the production of hydrogen peroxide, which is believed to be the principal virulence factor of the mycoides group of mycoplasmas. The genome also contains added watermark sequences for identification of the genome as synthetic and also antibiotic resistance markers to allow for selection.
Overlapping cassettes of 1080 were assembled from chemically synthesized oligonucleotides. 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 and transplanted in Mycoplama capricolum subspecies capricolum recipient cells in which the restriction modification system had been inactivated. This synthetic M. mycoides genome can be isolated for future work.
For sequence analysis of the M. mycoides sMmYCp235-1 genome, cells were grown in SP4 medium containing 10 mg/l tetracycline and genomic DNA was extracted using a Promega Genomic DNA Extraction Kit. Genomic sequencing via a small insert shotgun Sanger library generated 14319 successful reads. PCR and genomic walking for known repeats and problematic areas generated an additional 551 Sanger reads. The combined sequencing data was assembled using Celera Assembler (Meyers, 2000) resulting in 7 contigs in 3 scaffolds, with a contig N50 of 279215 bp. Average coverage was 11.5x. An additional 133 Sanger clone primer walks and genomic walks were generated to close the remaining gaps and cover low coverage regions. A completely finished genome was produced in 19 days.
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