The Seven Stones

SB 3.0 day 2

“Life, like a machine, cannot be understood simply by studying it and its parts; it must also be put together from its parts. Along the way to synthesizing a cell, we might discover new biochemical functions essential for replication, unsuspected macromolecular modifications or previously unrecognized patterns of coordinated expression.” (Forster and Church, 2006) .

The quest for generating a minimal cell may follow different paths, as illustrated by several talks presented at the Synthetic Biology 3.0 conference. Yesterday we heard from Giovanni Murtas how a purified transcription/translation system (36 recombinant enzymes + purified ribosomes) could be introduced into vesicles to successfully produce GFP. By extension incorporation of lipid synthesis enzymes, DNA replication enzymes and tRNA genes may enable the assembly of a minimal cell ultimately able to replicate both its core machinery and its envelope.

Converse to this assembly strategy, Hamilton Smith presented a top-down approach in which a genome is reduced to its minimum length by elimination of all non-essential elements, synthesized de novo and finally transfered into a host cytoplasm to boot a new cell. Mycoplasma genitalium appears to be a suitable organism for this type of experiments due to its small size (485 protein coding genes 43 RNA genes). Incidentally, M. genitalium also uses UGA (stop in E. coli) to code for Trp, which is a useful property to prevent expression of toxic products when fragments are assembled together via homologous recombination in E.coli. However, this requires in turn to use a Mycoplasma cell as acceptor cell. To set up the “transplantation” of naked DNA into host cell, the team tested the transformation of purified M. mycoides LC genomic DNA (via beta agarase digested agarose plug prep) into M. capricolum host (PEG-mediated transformation protocol). Nicely, the two genomes appear to segregate (how? incompatibility of origins of replication?) and various lines of evidence indicate that selected cells (tet resistance on donor DNA) contains DNA from the transplanted M mycoides genome only. Along similar lines, Rene Warren plans to rebuild H. influenzae using E. coli as a host. Without attempting to define a minimal genome, the idea is to assemble a series of 61 overlaping fosmids using the lambda red recombination system (Yu et al., 2000).

Assembly of 100 fragments via 4 rounds of homologous recombination, as described by Ham Smith, may appear as somewhat tedious and tricky. In a beautiful talk, Miroslav Radman showed how Deinococcus radiodurans manages to do this just by itself. It is almost spooky: after strong irradiation the genome of D. radiodiurans is chopped—literally pulverized—into small 20-30kb fragments. From this DNA soup, D. radiodiurans reassembles then a complete genome with high fidelity, within a couple of hours…. Apparently, small fragments are assembled via a synthesis-dependent strand annealing mechanism followed by larger fragment assembly via RecA-dependent homologous recombination (Zahradka et al, 2006). This process, a “molecular basis for resurrection”, was suggested by Miroslav to inspire the most imaginative projects, from “global sex” to “directed panspermia” and ressucitation of dead neurons…

see also: Brendan1 Brendan2 Brendan3 ETC blog

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