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Tinker, Tailor: Can Venter Stitch Together A Genome From Scratch?
Science, February 14, 2003

Craig Venter can't stand to be bored. No sooner had he and his team at Celera Genomics finished sequencing the human genome than Venter set another modest challenge for himself: He would tackle the world's environmental woes. His self-proclaimed goal (which landed him in newspapers and magazines around the world a few months ago) is to create microbes from scratch that can produce clean energy or curb global warming. To make this a reality, he set up a new organization, the Institute for Biological Energy Alternatives (IBEA) in Rockville, Maryland, right next to The Institute for Genomic Research that he founded in 1992. He got a small vote of confidence last November, when the Department of Energy awarded IBEA $3 million to take the first few steps toward that goal.

Venter predicts he will pull off the first step--creating a synthetic genome that, when inserted into a cell, can live and replicate--within 3 years. But experts on microbes and genomics are not so sure. No one has ever synthesized a string of DNA hundreds of thousands of base pairs long, much less "brought it to life." Obstacles range from determining which genes are essential to how to switch on a new genome. As for going the next step and creating a new pollution-fighting bug, many dismiss the scheme as science fiction or, at best, decades away. But Venter's critics and champions are closely watching what happens to the synthetic genome. "If he does make it, it will be a momentous achievement," promising insights into the fundamental workings of all living things, says Eugene Koonin, an evolutionary biologist at the National Center for Biotechnology Information in Bethesda, Maryland.

Step one

Venter's project has its roots in the mid-1990s, when he and his colleagues sequenced the peculiar genome of Mycoplasma genitalium, a species of bacteria that lives in the human urinary tract. They discovered that M. genitalium has only 517 genes, making it among the smallest genomes known (Science, 20 October 1995, p. 397). Its tiny size raised some fundamental questions, says Venter: "Is there a smaller set [of genes] we don't know about? Is there a way we can define life at a molecular level?"

To find out, Venter joined up with Clyde Hutchinson of the University of North Carolina, Chapel Hill, and other researchers to test whether individual genes were essential for M. genitalium's survival in the lab. They would knock out a gene, watch the microbe thrive or wither, and then repeat the experiment with a different gene. A surprising number of genes turned out to be superfluous, and Venter's group estimated that M. genitalium might be able to survive on only 265 to 350 genes (Science, 10 December 1999, p. 2165).

But testing one gene at a time could not tell the researchers exactly what would constitute such a minimal genome. It's possible, for example, that any one of five genes can do some vital task. Tested one at a time, all five may seem unnecessary, but if all of them are removed, the microbe dies. "We didn't have a way of doing cumulative knockouts to get down to just having a chromosome with just 300 genes in it," Venter explains. "So we don't know which are the right 300 genes, or even if 300 is the right number. That study drove my thinking that we needed a synthetic genome."

At IBEA, a team of a dozen scientists has been at work on the project for several months, led by Hamilton Smith, the 1978 Nobel Prize winner who has collaborated with Venter on the sequencing of many genomes. Their first challenge is daunting enough: to stitch together a string of nucleotides hundreds of thousands of base pairs long. The biggest genome synthesized to date is that of the 7500-nucleotide poliovirus (Science, 9 August 2002, p. 1016). "It's not trivial to do these syntheses," says Hutchinson.

Synthesizing DNA brings with it the small risk of introducing a typographical mistake into the code. "If you put thousands of these things together, then the chances that you can assemble something that doesn't contain any errors are very small," says Hutchinson.

Aniko Paul, a member of the team at the State University of New York, Stony Brook, that assembled the poliovirus last year, agrees: "Our project was technically much more difficult than we expected." She and her colleagues had to study each segment of the sequence carefully to make sure that it was error free.

Nor is it obvious which genes should be included in a synthetic genome. Microbiologists are familiar with the function of only a few well-studied microbes, such as Escherichia coli. Other microbes, such as M. genitalium, are still a puzzle. For instance, the function of a third of the genes that Venter's group has identified as potentially essential is unknown.

The researchers can't hope to construct every possible combination of M. genitalium genes and see which survives and which doesn't. There are simply too many possibilities. Venter believes that this brute force won't be necessary, citing intriguing evidence that bacterial genomes may be organized into functional groups ("cassettes") that all work together to do some particular job, such as metabolizing a specific molecule. Instead of testing every gene, Venter is banking on limiting the search to a much smaller set of cassettes.

Once Venter's team members have a full genome ready for a test drive, they will have to give it a home. They envision destroying the natural DNA of another M. genitalium and then inserting the synthetic genome into the microbe. What happens next is a mystery. "How do you boot up a new genome?" asks Bernhard Palsson of the University of California, San Diego, who builds computer models of the metabolism of microbes. "Now that you've made it, how do you get it going?" Paul and her colleagues didn't face this question with the poliovirus genome because a virus is not exactly alive. In essence, it's a genome that can hijack the cellular machinery of a living host. Bacteria, on the other hand, are truly alive, and their survival depends on the complex interactions of their genes and proteins. It's possible, Palsson suggests, that a synthetic genome might just lie inert in its new home, unable to spring to life.

"How do we know?" Venter responds. He points out that biochemists insert small sets of genes all the time into bacteria, which happily start using them to make new proteins. If a synthetic genome is properly designed, he argues, the machinery of a host microbe might automatically recognize it, and the microbe would start replicating on its own. "It wouldn't surprise me if it just happens," says Venter.

Biologists such as Palsson and Koonin would be delighted if Venter could get this far. "I've been talking for years with my colleagues about how we have to do this," says Palsson. For one, success would offer proof that there is such a thing as a minimal genome. "Ultimately, proof by synthesis is the most convincing way to demonstrate that we understand the basic principles of any process," says James Shapiro, who studies microbe evolution at the University of Chicago. A microbe with a synthetic genome could offer more than a proof of concept, however. It could serve as a sort of microbial fruit fly that biologists could use to investigate basic aspects of life itself.

Step two

But Venter has grand plans for his "minimal microbe." He hopes to use it as a foundation for building a much larger genome, endowed with new genes that would enable it to produce hydrogen fuel on an industrial scale or efficiently suck in the carbon dioxide released by a power plant. "If he can do it, more power to him--but I think it's going to be really hard," says Suellen van Ooteghem of the National Energy Technology Laboratory in Morgantown, West Virginia. Others question the need for a new microbe, should it be possible to create one. But Venter, with characteristic brashness, believes he can improve on evolution.

Even if the team successfully builds a genomic skeleton, it would have to add a lot of muscle--perhaps 1000 additional genes--to enable it to fight pollution. Venter and colleagues plan to scour nature for the most powerful combination of genes. But figuring out how to add the regulatory functions to ensure that all these genes turn on and off at the right time will be difficult, if not impossible. "Our understanding of regulatory circuits, even in bacteria, is actually quite poor," says Koonin.

Venter recognizes the profoundness of the challenge he has set for himself and his co-workers. "There are so many unknowns, and no one's been there before," he says. "All this is pure basic science, and we're learning as we go." But that hasn't stopped Venter before, and it doesn't seem to be stopping him now.

Copyright © 2002 Carl Zimmer. Reproduction or distribution is prohibited without permission from the author.

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