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Yeast Construction Zone

By building the first synthetic yeast genome, Hopkins researchers hope to create a versatile biotech tool for such tasks as producing drugs or new types of fuel.

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Image by Mike and Amanda
Knowles

November 2010-- In 2002, scientists produced the first synthetic viral genome, an advance that ushered in a new field of science, called synthetic biology. Since then, synthetic biologists have accomplished more sophisticated feats—the synthesis of an even larger viral genome and, this past spring, the construction of the first artificial bacterial genome. 

Now a research team at Hopkins is aiming to achieve an even larger goal in this new discipline, to build the first synthetic yeast genome.

Why attempt this feat? The phrase “because it’s there” comes to mind. And Jef Boeke, professor of Molecular Biology and Genetics, is the first to admit that the project “does have a Mount Everest aspect.” Now that biologists have synthesized the genomes of viruses and a bacterium, it’s time to scale even higher heights. With a synthetic yeast genome, scientists will have built the first artificial eukaryotic genome, the biological category that includes humans and other organisms whose cells have membrane-enclosed nuclei and other complex structures.

But that conquest isn’t the team’s sole motivation. Another is basic knowledge. “We want to understand the function of each piece of the yeast genome,” says Joel Bader, an associate professor of Biomedical Engineering who, along with Boeke and Srinivasan Chandrasegaran, a professor of Environmental Health at the Johns Hopkins Bloomberg School of Public Health, is directing the project. And by harnessing that knowledge, scientists may be able to design yeast to do certain jobs, such as produce protein-based drugs or generate biofuels.


12 million pieces

To synthesize a genome, scientists use published sequence information for that organism—the order of the four nucleotide bases (adenine, thymine, guanine, and cytosine) that compose DNA. That sequence provides the instructions for producing and piecing together the proteins and other molecules needed to build the particular organism.

But synthesizing the yeast genome is far more laborious than the handful of other genomes generated to date. Poliovirus, the first viral genome synthesized, is just 7,500 nucleotide bases long. The first synthetic bacterial genome, a copy of Mycoplasma mycoides, has just one chromosome containing 1.08 million nucleotide bases. Yeast, or Saccharomyces cerevisiae, in contrast, has 16 chromosomes containing 6,000 genes, and is 12 million nucleotides in length.

How to go about such a daunting feat? At first, Boeke thought he would work with industry. He formed a partnership with a biotech company and asked it to synthesize 100,000 base pairs of the yeast genome. That project took a full year, and Boeke realized that the cost and time required to synthesize the entire genome that way would be prohibitive.
Then one day he had a thought: “Wait a second. There are all these Hopkins undergraduates who want a meaningful lab experience. We could recruit them to be our army that makes small pieces of yeast DNA that get assembled to make synthetic chromosomes.”

That lightbulb moment gave birth to the Build-a-Genome course, which Boeke, Chandrasegaran and Bader have taught for seven semesters. After learning basic techniques during a month-long boot camp, each student is given keys to the wet lab and assigned 10,000 base pairs or more of the genome to synthesize. As segments of the genome are assembled, the researchers then install them in yeast cells in place of the corresponding bits of native DNA.


Not a carbon copy

The scientists do not plan to faithfully follow Nature’s recipe for yeast. Instead, they are systematically re-engineering the genome in several ways.

First, the scientists are, in effect, cleaning house. Evolution has shaped and fine-tuned the yeast genome. Yeast are good at what they do, as any baker or brewmaster can attest. But scientists have also found much extraneous matter and redundancy within yeast’s DNA. So the Hopkins group is identifying and disposing of these nonessential bits.

“We’re making yeast tidier,” says Boeke, “and more neat, trim and stable.” Removing such extraneous code reduces the scope of the synthesis task and will allow researchers to test just how small and efficient a yeast genome can be and still function—a first step toward a highly efficient tool for generating useful proteins.

Then, the team is also developing a technique that will enable it to change the genome in myriad ways, by inserting a special enzyme called a recombinase into the synthetic genome. The recombinase will cut and rearrange segments of DNA throughout the genome, in effect shuffling genes around the way one might shuffle a deck of cards. Only this mechanism will also sometimes drop a card, or gene, from the deck, and sometimes the process will also duplicate certain genes. The result: billions upon billions of possible variations on the theme of the yeast genome. (Not all of the variations will be viable, says Bader.)

“The shuffling will help us learn which genes are required for different conditions,” says Bader. “And we’ll be able to see whether yeast can compensate for losing some genes by over-copying others.” The technique will also give scientists a versatile tool that they can use to select or screen for yeast genomes specialized for certain tasks. For example, engineers would like to be able to turn straw, wood and other plant material into a biofuel called cellulosic ethanol. Yeast might be the factories for performing this step, except they normally do not like to grow in such crude fuel sources. But genome shuffling might yield a variety of yeast that can grow and even thrive in such an environment.


Ethical issues

Why to synthesize a genome and how to do it aren’t the only questions raised by this and other synthetic biology projects. Another is whether to do it at all: Should scientists synthesize an artificial yeast genome—or any genome? What limits and regulations should be placed on such research? Some critics question whether scientists who synthesize genomes are playing God or at least reenacting the role of Dr. Frankenstein. Boeke says he would be opposed to synthesizing a human genome for the purpose of reprogramming a human being but has no qualms about synthesizing the genome of yeast or other nonpathogenic microbes.

However, he notes that synthesis of even a microbial genome raises certain safety issues that warrant discussion. Debra Mathews, a bioethicist at the Johns Hopkins Berman Institute of Bioethics, raises such issues in a lecture she delivers to students in the Build-a-Genome course.

Mathews says she asks students to think about the “unknowns” associated with synthetic biology. For example, if scientists synthesize a new oil-eating microbe, they won’t know exactly how it will affect the environment until they take it out of the lab and use it in an environment. There’s also the issue of dual-use technologies: the idea that someone could exploit a synthetic genome for nefarious purposes, such as altering a drug-producing synthetic yeast so that the organism produces a toxin instead.

“We don’t know how easy it would be to create such ‘super bugs,’” says Mathews. But future scientists should be contemplating the implications of the techniques that they are learning.

Given the work still remaining on the yeast synthesis project (only about 10 percent of the genome has been completed), students and their mentors have several more years to discuss and debate those implications before the world receives its first yeast with a genome designed and built in a lab. 

–Melissa Hendricks

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