M. genitalium Genome Made From Scratch By JCVI Scientists

"The actual synthesis and assembly of this genome presented a formidable technical challenge," said Daniel G. Gibson, lead author of the team's recently published Science article, "because as strands of DNA get longer the get increasingly brittle." To overcome the risk of breakage, JCVI scientists built the artificial chromosome from 5,000 to 7,000 base-pair long chemically-synthesized DNA fragments that were assembled into 101 "cassettes" and outsourced to Blue Heron, DNA 2.0 and GENEART for production. The cassettes, each containing one or more complete genes, were then stitched together into larger and larger pieces and eventually combined into an entire synthetic M. genitalium genome.

Initially, sets of four cassettes were joined to create 25 subassemblies, each about 24kb (24,000 base pairs) long. The fragments were first cloned as bacterial artificial chromosomes (BACs) in Escherichia coli, which produced enough DNA for the next steps as well as for DNA sequence validation. However, E. coli proved incapable of producing half-genome clones and the group turned to the yeast Saccharomyces cerevisiae to complete the job. Several years of work had shown JCVI researchers that homologous recombination in S. cerevisiae could build an entire bacterial chromosome from large subassemblies. Indeed, S. cerevisiae surpassed the scientists' greatest hopes by assembling and then cloning the one-quarter cassettes into the final M.genitalium genome in a single step. Nonetheless, they caution, "we don't as yet know how generally useful yeast will be as a recipient for bacterial genome sequences."

Strategically placed DNA segments serving as "watermarks" enabled the scientists to confirm the assembled genetic material was synthetic and not native. But the watermarks are also a source of amusement for J. Craig Venter, who assembled the genome-synthesizing team, because they contain encrypted messages can be deciphered by determining their amino acid sequences. "It's a fun thing that has a practical application," he says.* Additionally, a 2,514 bp insertion, containing an aminoglycoside resistance gene, was embedded in gene MG408 (msrA), both to block pathogenicity and allow for selection.

Drew Endy, Assistant Professor of Biological Engineering at MIT calls the work "a great technical feat and demarcation point for important changes in both genetics and biological engineering." Citing the "Carlson Curves" (DNA sequencing technology is improving exponentially, doubling every 12-18 months), Endy says he "wouldn't be surprised if the genomes of bacteria and single-celled eukaryotes were being routinely designed and constructed by 2012." However, although he admires the technical achievement, Rob Carlson, the physicist turned molecular scientist who formulated the Curves, points out that the JCVI team was unable to successfully boot up the new organism because of a technical glitch. The team discovered the problem —a TARBAC vector that interrupted a small gene— while their paper was in press. The vector has been moved, but a number of other technical barriers to the development and transplantation of synthetic chromosomes remain, they say.

The next, more difficult, task facing JCVI scientists is creation of a living bacterium with an entirely synthetic genome. J. Craig Venter predicts this will happen sometime within the year. JCVI researchers have already shown that whole genome transplantation is technically feasible by converting one species of Mycoplasma into another. (See Microbe , Vol.2, October 2007.) Venter says that a green jet fuel is now being produced by metabolically-engineered designer cells at the privately-held Synthetic Genomics Inc. and he expects hundreds of other products to follow. Some of these products, he hopes, will soon be made by synthetic cells with laboratory-designed metabolic cassettes. Notably though, Hamilton O. Smith, who leads the microbiology teams at both Synthetic Genomics and JCVI, considers it unlikely that M. genitalium will ever be employed as a designer organism in such commercial endeavors because "it's so difficult to grow in the lab."

A full description of the JCVI research described above can be accessed online in Science, 24 January 2008; 10.1126/science.1151721.