A few weeks ago, Jason Kelly explained in his post how Itaya and colleagues (2007) assembled the complete 135 kb rice chloroplast circular genome starting from a collection of 5-6 kb fragments and using sequential in vivo homologous recombination in Bacillus subtilis. Now, Hamilton Smith, Craig Venter and colleagues have achieved the assembly of a complete 583 kb Mycoplasma genitalium genome (“
JCVI-1.0”, Gibson et al, 2008). The starting fragments were of similar length, 4-5 kb fragments with 80-360 kb overlaps, albeit synthesized chemically rather than by PCR. In contrast to Itaya et al, Ham Smith’s team used in vitro recombination (using T4 pol digestion/annealing/Taq pol repair and ligation) in a 3 step hierarchical assembly process and completed the fourth step, the assembly of 4 quarter genomes, using in vivo homologous recombination in yeast (TAR cloning, Larionov et al, 1996). The use of yeast for the last step might be a little worrying, given the high recombination activity in yeast and the propensity for large constructs to rearrange (I used to work with YACs to construct mouse transgenes and I can still feel the pain… but I don’t know about the stability of circular TAR clones). In any case, it worked! One final clone was sequenced (7X coverage) and, remarkably, was shown to match exactly the sequence designed!
This impressive technical feat may eventually have tremendous consequences when combined with the transformation procedure (“genome transplantation”, ) Venter and colleagues reported last year (Lartigue et al, 2007). As Dawkins noted at the Digital Life Design meeting in Munich a few days ago (see video below for some excerpts of his discussion with Craig Venter and the transcript in Edge), “genetics has become a branch of information technology”.
JCVI-1.0 has obviously not been assembled “from scratch”. In fact, beside some “watermark” sequences inserted to distinguish the synthetic genome from the native one, the fact that its sequence is a remarkably accurate copy of M. genitalium genome is probably one of the major achievements of the study. The technology for the synthesis of very long DNA of arbitrary sequence (in principle…) is thus progressing at an impressive pace. But writing a genome is not (yet) equivalent to designing it. Exciting (and hard) work remains to be done to bridge this gap and to improve our understanding of how biological functions can be created by assembling genes into a synthetic genome and developing the tools that will make this process rational and efficient, a challenge the synthetic biology community is eager to tackle (see The BioBricks Foundation)…