RESUMO
We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication.
Assuntos
Bioengenharia , Engenharia Genética , Genoma Bacteriano , Mycoplasma capricolum/genética , Mycoplasma mycoides/genética , Proteínas de Bactérias/análise , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/síntese química , DNA Bacteriano/genética , Escherichia coli/genética , Deleção de Genes , Genes Bacterianos , Dados de Sequência Molecular , Mycoplasma mycoides/crescimento & desenvolvimento , Mycoplasma mycoides/fisiologia , Mycoplasma mycoides/ultraestrutura , Fenótipo , Plasmídeos , Reação em Cadeia da Polimerase , Polimorfismo Genético , Saccharomyces cerevisiae/genética , Transformação BacterianaRESUMO
Most microbes have not been cultured, and many of those that are cultivatable are difficult, dangerous or expensive to propagate or are genetically intractable. Routine cloning of large genome fractions or whole genomes from these organisms would significantly enhance their discovery and genetic and functional characterization. Here we report the cloning of whole bacterial genomes in the yeast Saccharomyces cerevisiae as single-DNA molecules. We cloned the genomes of Mycoplasma genitalium (0.6 Mb), M. pneumoniae (0.8 Mb) and M. mycoides subspecies capri (1.1 Mb) as yeast circular centromeric plasmids. These genomes appear to be stably maintained in a host that has efficient, well-established methods for DNA manipulation.
Assuntos
Clonagem Molecular/métodos , Genoma Bacteriano , Mycoplasma/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Diploide , Vetores Genéticos/química , Dados de Sequência Molecular , Mycoplasma genitalium/genética , Mycoplasma mycoides/genética , Mycoplasma pneumoniae/genética , Recombinação GenéticaRESUMO
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.