Literature

Craig Venter has made a big impact with his announcement of assembling a synthetic version of the Mycoplasma genitalium genome. The work started many years ago when the bacterium with the smallest genome (M. genitalium) was sequenced. The research team then set out to make it artificially.

They "designed fragments of chemically synthesized DNA to build 101 "cassettes" of 5,000 to 7,000 base pairs of genetic code. As a measure to differentiate the synthetic genome versus the native genome, the team created "watermarks" in the synthetic genome. These are short inserted or substituted sequences that encode information not typically found in nature. Other changes the team made to the synthetic genome included disrupting a gene to block infectivity. [. . .] From here, the team devised a five stage assembly process where the cassettes were joined together in subassemblies to make larger and larger pieces that would eventually be combined to build the whole synthetic M. genitalium genome. In the first step, sets of four cassettes were joined to create 25 subassemblies, each about 24,000 base pairs (24kb). These 24kb fragments were cloned into the bacterium Escherichia coli to produce sufficient DNA for the next steps, and for DNA sequence validation. The next step involved combining three 24kb fragments together to create 8 assembled blocks, each about 72,000 base pairs. These 1/8th fragments of the whole genome were again cloned into E. coli for DNA production and DNA sequencing. Step three involved combining two 1/8th fragments together to produce large fragments approximately 144,000 base pairs or 1/4th of the whole genome."

The watermarks have been decoded quickly, as a report in Wired Science reveals. The words are:
VENTERINSTITVTE
CRAIGVENTER
HAMSMITH
CINDIANDCLYDE
GLASSANDCLYDE
The latter three words refer to Venter's colleagues. The reporter commented: "I have to say that I was hoping for something poetic, profound, or clever. [. . .] Instead, we get what we probably should have expected: an advertisement for the Venter Institute."

The quest for artificial life

Large fragments of DNA are very difficult to handle, and this delayed completion. As Philip Ball explains:

"for reasons that the researchers don't yet understand, the final assembly of these quarter-genomes into a single strand didn't run smoothly in the bacterium. So the team transferred them into cells of brewers' yeast to carry out the final steps of the assembly. Smith and his colleagues then extracted the synthetic genomes from the yeast cells, using enzymes to chew up the yeast's own DNA."

The final product has 582,970 base pairs. The assembled genome was again sequenced in order to validate the accuracy of the chemical structure.
By all accounts, this is a remarkable achievement. It has been described as "a technical tour de force" and a "monumental effort". The authors are obviously proud of their success:

"The actual synthesis and assembly of this genome presented a formidable technical challenge. Although chemical synthesis of genes has become routine, the only completely synthetic genomes so far reported have been viral. The largest previously published synthetic DNA that we are aware of is a 32 kb polyketide gene cluster."

Much can be said about the significance of this research. We will limit comments here to address just three points.

1. Whilst the team have successfully assembled the genome, they have had to get some assistance from the humble yeast Saccharomyces cerevisiae. Long strands of DNA were found to be very fragile, and final assembly needed to be done inside yeast cells. Whilst the process is understood to be "transformation-associated recombination", the researchers were not able to do this artificially. It needed the environment of a cell to make it happen. The difficulties of working with DNA strands should promote some appreciation of why intelligent design is so relevant to biology. Those who continue to claim that life assembled as a bottom-up process are not rooting their thinking in the real world.

2. The next phase of research, according to Venter and his team, is to move from the genome to the functioning cell. The new research aim is to discover whether cells can be 'booted up' into action when loaded with this genetic programme. "This is the next step and we are working on it" said Smith (coauthor). Once they can do this, they have the tools to experiment with different genomes: knocking out particular genes and discovering the consequences, and engineering new genes to achieve specific outcomes. Last year, the research team reported a technique for replacing M. genitalium's genome with another taken from a different species, so it might be thought this step is not a major one. However, there are big questions about what is really going on. What is the nature of the interaction between the cell and the genome? Does the cell have information embedded within it that is as important, if not more important, as the digital DNA information? These questions are being asked by people who have not bought into the popular genetic reductionism that has become dominant in this research arena.

3. There are ethical concerns about this research. Listening to Craig Venter today in a radio interview, it was clear he wanted to give reassurances that the scientists really know what they are doing and that unforseen things are not going to happen. The Science Daily report says: "The bioethical group's independent deliberations, published at the same time as the scientific minimal genome research, resulted in a unanimous decision that there were no strong ethical reasons why the work should not continue as long as the scientists involved continued to engage public discussion." Despite all this, some of us remain unconvinced that the researchers really know what they are doing. We are concerned because of the prevalence of genetic reductionism. We are not saying that the researchers are devious people, but they do need to display a more holistic perspective on the workings of living things. They appear to think that if they can control the genome, they control everything. It is this latter view that can lead to dangers being overlooked and safeguards being inadequate.

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 Express, January 24, 2008 | DOI: 10.1126/science.1151721

We have synthesized a 582,970 bp 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 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 (BACs) 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 (TAR) 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.

See also:

Ball, P. Genome stitched together by hand, news@nature.com, 24 January 2008, doi:10.1038/news.2008.522

Scientists Create First Synthetic Bacterial Genome - Largest Chemically Defined Structure Synthesized In The Lab, ScienceDaily (January 24, 2008)