Life in the lab, creating, chemically!

D Balasubramanian, in his latest column in the Hindu explains some of the details:

They chemically synthesised many fragments of the DNA, encoding the 582,970-units-long genome of a bacterium called M ycoplasma genitalium. Next, they assembled these fragments in perfect order to generate the genome of the bacterium.

The DNA sequence of the synthetic one was confirmed to be identical to the natural one. Preliminary results were first published in the 24 Jan 2008 issue of Science, and the more detailed successful one in the 5 Dec 2008 issue of PNAS (US).

The task is somewhat akin to writing phrases and sentences first, and then putting them together in proper order to make the meaningful chapter of a book.

While the DNA pieces were synthesised chemically, the stitching together was done using the biochemical machinery of a host cell. About 100 pieces of the genome, each 5000-7000 units long in DNA sequence, were first joined together to produce 25 sub-assemblies, each about 24000 base pairs long.

These were then introduced into the bacterium E. coli to produce sufficient DNA for the next steps. Next, they repeated the procedure to generate large fragments comprising 1/4th of the whole genome of M genitalium.

Now, they used the clever trick of exploiting the process called homologous recombination. This is a basic essential process in every cell, which physically rearranges the two strands of DNA.

It involves the alignment of similar sequences, is used in DNA repair and to restart replication that has been stalled or damaged. And an organism like yeast does this more efficiently, wholesale faster than bacteria like coli.

The JCVI researchers inserted the synthesised DNA fragments into yeast and utilised its homologous recombination ability to generate the whole 580,000 long genome of M genitalium in one step. As one of the authors of the paper remarked: “I am astounded by our team’s progress in assembling large DNA molecules. It remains to be seen how far we can push this yeast assembly platforms to boot up the synthetic chromosome”.

This is clearly a landmark work that leads into the brave new world of synthesising life itself in the laboratory. It was hardly 200 years ago when Friedrich Wohler synthesised urea, an organic molecule, in the chemical laboratory, thus throwing out the notion of “vital forces” involved in the components of living organisms.

Balasubramanian goes on to conclude the piece with some discussion on the ethics and morals of the research after making an exciting prediction:

If only we find a way to insert the bacterial genome into this proto-cell, and somehow trigger it (electric pulse? ionic currents?) to make the bacterium itself!! We would have chemically created life in the lab. This is not a pipedream; JCVI scientists are already on the job, and my bet is they will do it within a few years.

A wonderful piece; don’t miss it.

PS: I think Balasubramanian gets both the references wrong though — there is no 24 January issue in 2008, and no PNAS issue on 5th December; But, I believe the references are to the following two papers:

[1] Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome,
Daniel G. Gibson, Gwynedd A. Benders, Cynthia Andrews-Pfannkoch, Evgeniya A. Denisova, Holly Baden-Tillson, Jayshree Zaveri, Timothy B. Stockwell, Anushka Brownley, David W. Thomas, Mikkel A. Algire, Chuck Merryman, Lei Young, Vladimir N. Noskov, John I. Glass, J. Craig Venter, Clyde A. Hutchison, III, Hamilton O. Smith, Science, 29 February 2008, Vol. 319. no. 5867, pp. 1215 – 1220.

Absract:

We have synthesized a 582,970–base pair Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted “watermarks” at intergenic sites known to tolerate transposon insertions. Overlapping “cassettes” of 5 to 7 kilobases (kb), assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb (“1/8 genome”), and 144 kb (“1/4 genome”), which were all cloned as bacterial artificial chromosomes in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments.

[2] One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome,
Daniel G. Gibson, Gwynedd A. Benders,Kevin C. Axelrod, Jayshree Zaveri, Mikkel A. Algire, Monzia Moodie, Michael G. Montague, J. Craig Venter, Hamilton O. Smith, and Clyde A. Hutchison III, PNAS, Published online on 10, December 2008 before print.

Abstract:

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.

The PNAS piece is an Open Access article too. Have fun!

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