By Kevin E. Noonan --
Craig Venter's colleagues at the J. Craig Venter Institute show no signs that vocal criticism of their work in synthetic biology has made a dent in their determination to "create life" (see "Playing the Bioterror Card in the Synthetic Biology Debate"). They report in this week's Science Express (an online journal of pre-publication posted papers) that by successfully chemically synthesizing an entire bacterial chromosome, they have taken the next step in their efforts to create "life" in a test tube (see Gibson et al., "Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome," abstract - full version of article available to subscribers only).
The bacterial chromosome in question comprises about 385 "essential" genes from the 485-gene Mycoplasma genitalium genetic complement (see below). The researchers had determined the identity of the 100 "non-essential" genes using conventional genetic techniques, but are not yet certain whether these genes can all be deleted simultaneously. The synthesis reported this week is directed at making that determination.
The synthesis was not completely synthetic, in the sense that the final chromosome was not produced solely from in vitro chemical synthesis. Rather, chemically-synthesized oligonucleotides of 5 to 7 kilobases in length were ligated to produce intermediates of 24, 36, 72 and 144 kilobases. These intermediates were cloned into bacterial artificial chromosomes (BACs) and sequenced, and then the final chromosomes were assembled by transformation-associated recombination in yeast (S. cerevisiae) using yeast artificial chromosome vectors (YACs). This final assembly was isolated and clones with the correct sequence identified. The size of the completed genome was 582,970 basepairs (about 3 kb larger than the native bacterial chromosome).
This synthetic chromosome also contained what the researchers termed "watermarks," insertions of transposable element sequences at intergenic sites. These markers were diagnostic for the artificial chromosome, as was the insertion of an antibiotic resistance element in a gene (MG408) that acts as a virulence factor, thus in one step permitting selection of the artificial chromosome and eliminating human pathogenicity.
This work complements earlier work where the group was able to transplant the genome of one Mycoplasma species into another (see links below). It sets the stage for determining which of the 100 non-essential genes can be deleted simultaneously, and for the project's ultimate goal of complete synthetic reconstruction of a living organism.
For information regarding this and other related topics, please see:
- "Playing the Bioterror Card in the Synthetic Biology Debate," December 19, 2007
- "The Synthetic Biology Sky Is Not Falling," December 16, 2007
- "Patenting Life (Part II)," June 29, 2007
- "Patenting Life (Really)," June 11, 2007
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