Saturday, January 26, 2008

Entire Synthetic Genome Created

John Roach for National Geographic News
January 25, 2008

Scientists yesterday announced that they have successfully created an entire synthetic genome in the lab by stitching together the DNA of the smallest known free-living bacterium, Mycoplasma genitalium.

Experts are hailing the research as an important breakthrough in genetic manipulation that will one day lead to the "routine" creation of synthetic genomes—possibly including those of mammals.

This is "a striking technical accomplishment," biochemist Leroy Hood, who was not involved in the study, wrote in an email.

"It represents the initial stages of an important new step in studying how genes function together in systems to create complex phenotypes [traits]," added Hood, co-founder of the Institute for Systems Biology in Seattle, Washington.

Step Toward Artificial Life

The new work is an important second step in a three-step process to the creation of synthetic life, said research leader Hamilton Smith, a biologist and Nobel laureate at the J. Craig Venter Institute in Rockville, Maryland.

The first step, reported last year by the same team at Venter's institute, was the successful transplantation of a genome from one species of bacteria into another, effectively switching the bug's identity.

"The third step, which we're working on now, is to take the chemically synthesized DNA, which is in the test tube, and get it into a bacterium where it can take over and produce a synthetic cell," Smith said.

Researchers liken that step to rebooting a computer, because a genome is akin an operating system that makes a cell function.

Successfully completing the final step would create the first synthetic life-form.

The research is also part of a project to create a cell with "the smallest number of genes that can still confer life," Smith added.

The team chose M. genitalium because it contains about 485 genes, the smallest known of any organism capable of surviving on its own. The researchers suspect that about 400 genes are necessary for life.

Once they have created a synthetic copy of the bacteria, scientists can begin to eliminate genes to determine which are essential.

Such an accomplishment would then allow scientists to create synthetic life-forms that may one day produce biofuels, clean up toxic waste, and fight global warming. (Related: "Gene-Altered Plant, Tree Can Suck Up Toxins" [October 15, 2007].)

"So, this is only the beginning," Smith said.

Genome Assembly

But completing that second step was no easy feat.
While scientists can pretty easily assemble short sequences of DNA—or order them out of a catalog—synthesizing entire genomes is difficult.

That's because as more base pairs of the four building blocks of DNA—adenine, cytosine, guanine, and thymine—are stitched together, the strands tend to weaken and eventually break.

Prior to this research, for instance, the longest synthesized string contained 32,000 base pairs of DNA. The M. genitalium genome is 582,970 base pairs.

So the researchers broke up the genome into 101 segments, called cassettes, each containing between 5,000 and 7,000 base pairs of genetic code.

The researchers also took steps at this stage to address concerns that the technology could be misused to engineer a deadly virus or that an unforeseen innocent error could lead to bacteria run amok.

The researchers added watermarks to the code to differentiate the synthetic DNA from genomes of wild M. genitalium. They also inserted a gene to block the ability of the synthetic genome to infect human or animal hosts.
Much of the cassette assembly work was outsourced to genetics companies.

Back at the lab, meanwhile, Smith and his colleagues devised a process to stitch the 101 cassettes into a full synthetic genome.

They combined increasingly larger sections of the genome together in a test tube with linking and repair enzymes found in the bacterium Escherichia coli until they had four overlapping quarter genome sections.

After unsuccessfully trying to combine the quarters into halves in E. coli, the team switched to brewers' yeast, and the genome came together through a process the yeast uses to repair damaged DNA.

"That was pretty remarkable," Smith said, explaining that scientists had not known that a single yeast cell would pick up all the overlapping pieces and correctly assemble them.

"Yeast will play a big part in the future in assembling large DNA molecules," he added.

Smith and his colleagues report the assembly process in a paper published on the Web site of the journal Science.

"Important Changes" to Genetics

Drew Endy is an assistant professor in the department of biological engineering at the Massachusetts Institute of Technology in Cambridge and an expert in the field of synthetic biology. He was not involved in this study.

In an email, he said "reconstructing a natural bacterial genome from scratch is a great technical feat."

While genomes up to eight million base pairs have previously been assembled from existing DNA fragments, he noted the new accomplishment heralds "important changes in the science of genetics."

By 2012, he added, the technology should exist to routinely design and construct genomes of any bacteria or single-celled organism with a membrane-bound nucleus.

"Which also means," he said, "that it will be possible to construct some mammalian chromosomes."

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