PETER AND MARIA HOEY
In the late 1990s, a handful of physicists and engineers began to take a greater interest in biology. The Human Genome Project was spitting out more and more gene sequences—blueprints for the protein building blocks of the cell—generating a flood of new information about the molecular machinery of life. Trouble was, there were not enough biologists doing the job of figuring out how all these genes and proteins worked together to create a living, breathing organism.
It was around this time that Boston University bioengineer James Collins saw his chance to inject a little engineering know-how into the study of biology. There were two ways to go about it, he figured—either disassemble cells or build them. “A burgeoning young engineer [is] either the kind of kid who takes stuff apart to try to figure out how it works, or [he’s] the kid who puts stuff together,” Collins says. Though both approaches seemed promising, there simply wasn’t enough known about the structures or functions of the genes and their protein products to infer how all the parts worked together by taking a cell apart, piece by piece.
“Reverse engineering seems to be too challenging,” Collins recalls musing to his then grad student Tim Gardner. “But can we do forward engineering? Can we take parts from cells and put them together in circuits, just as an electrical engineer might?”
As the field works to create new living systems that serve a purpose...a new foundation for biological understanding should emerge.
—Pamela Silver, Jeffrey Way, “Cells by Design,”The Scientist, September 27, 2004
The answer was yes. After two years of tweaking various characteristics of transcriptional repressors in E. coli, the team succeeded in constructing biology’s first synthetic toggle switch—two repressor genes controlled by two promoters that caused their respective repressors to be expressed by default. The repressors were designed to inactivate each other, however, such that the two genes would never be fully expressed at the same time. The addition of a stimulus, such as a chemical pulse to suppress one gene long enough for the other to come on, allowed the system to flip from one stable state (gene A on, gene B off) to its other stable state (A off, B on).
The results were published in 2000, alongside a paper from physicist Stanislas Leibler’s lab at Princeton University, which had undertaken a similar, but independent, project. Much like Collins with Gardner, Leibler teamed up with his graduate student Michael Elowitz to build an oscillator, which, like Collins’s toggle switches, used transcriptional repressors in E. coli. The Princeton team engineered three genes to inhibit each other in a cyclical manner, rock-paper-scissors style, with each gene repressing the next when a threshold concentration of its gene product had been reached. The result was the periodic expression of all three genes—monitored by the periodic glow of green fluorescent protein (GFP), whose expression was linked to another copy of a promoter controlling one of the three repressors.
The two publications are now widely cited as the seminal papers of synthetic biology, though neither paper received much publicity at the time. “[We were] kind of a ragtag group of engineers and physicists who were essentially amateurs in molecular biology,” Collins says. But in the last decade, many trained molecular and cell biologists have turned to syn bio, designing synthetic circuits built from biological components and branching out from the transcriptional regulation tools of Leibler, now at Rockefeller University, and Collins to add translation and post-translation components.
The methods for actually manufacturing the circuits have also improved. While the Collins and Leibler teams were essentially cutting and pasting existing genes, J. Craig Venter and his colleagues went for a ground-up approach. They took the blueprint of a known bacterial genome and rebuilt the entire sequence, stitching together genes chemically manufactured by an automated DNA synthesizer. The genome was then inserted into the nucleus of another bacterium, with May 2010 headlines announcing the creation of the first cell to run on a genome synthesized entirely from scratch. (Read Venter’s opinion piece, "Synthesizing Life.")
Many researchers still use the basic cut-and-paste approach, however, employing well-vetted and still advancing genome-editing technologies to select different bits of DNA, called BioBricks, from living organisms and piece them together to form novel circuits. Others, like George Church of Harvard University, fall somewhere in the middle, synthesizing individual genetic components using oligonucleotide chips, then piecing them together. “I think it’s an open question as to whether the core of synthetic biology is going to make things by BioBricks, by total synthesis, or from scratch from chips in a modular way,” says Church.
Regardless of how the circuits are assembled, engineered organisms hold potential in a wide range of fields, including biofuel production, agricultural innovation, and biomedical advances. One of the most successful medical applications has been the engineering of yeast to produce a precursor of the antimalarial drug artemisinin, a natural product of the plant Artemesia annua. The production of the drug is currently limited to small farms in Southeast Asia, where farmers grow the plants and extract the drug using relatively crude techniques, making the drug expensive and often in short supply—a bad combination for the developing nations that need it most.
To address these problems, Jay Keasling of the Lawrence Berkeley National Laboratory and his colleagues decided to rebuild the artemisinin pathway in a more manageable microbial system. After several years of tweaking the molecular components first in E. coli, then in yeast, the researchers succeeded in building a synthetic circuit in yeast cells that generates a healthy supply of artemisinic acid—an artemisinin precursor. “If you were to take something like a 100,000-liter fermenter, and grow up our artemisinin-producing yeast, running that full time you could probably get enough artemisinin for the entire world,” Keasling says. With funding from the Bill & Melinda Gates Foundation and partnerships with California-based biotech Amyris, the Institute for OneWorld Health, and pharmaceutical giant Sanofi to optimize and scale up production and distribute the product to Africa, Keasling and his colleagues expect that the yeast-derived artemisinin will be commercially available by the end of this year, and that drugs containing the product will hit the market in early 2012.
Another synthetic biology inspired malaria project aims to stop transmission of the disease at the level of its vectors by engineering a genetic system to establish itself in a mosquito population. While researchers have successfully engineered mosquitos to be resistant to infection by the malaria parasite, introducing those mosquitos into the wild is not likely to result in sufficient spread of the resistance, as the wild-type genes will vastly outnumber the introduced variety. Something, such as a significant fitness advantage, must help drive the new genes into the population. Geneticist Bruce Hay and his team at Caltech got their inspiration for a solution to this problem from the Medea toxin/antidote genetic element in Tribolium beetles, in which a toxic maternal gene product kills any embryos that do not inherit the element, ensuring its quick spread through the population. Armed with 50+ years of Drosophila genetics knowledge, the researchers created a genetic element, Medeamyd88-1, which caused mother flies to produce eggs that only survived if they received a copy of the element.
In laboratory tests, Medeamyd88-1 quickly spread through the population, such that every individual carried the element by the 12th generation. Hay’s group is now working on developing a similar system in disease-carrying mosquitos. If he succeeds, “then it becomes a question of can we link these two pieces of biology together,” Hay says—the gene that makes the mosquitos disease-resistant and the Medea element that drives it through the population.
As was the intention of some of the field’s founding engineers, synthetic biology also promises to help researchers understand the basic rules of cellular function in ways that traditional biology hasn’t been able to, says Elowitz, now a professor at Caltech. “With the synthetic approach, you can start to think of the cell as a laboratory where you can tinker around and really ask questions about the basic principles of genetic circuit design.”
The growing influence of engineering in biology is, in some sense, “the best of both worlds,” adds Church. (See his opinion, "Evolving Engineering.") The good design principles of engineering and the unique properties of evolving biological systems are “just an incredible combination,” he says.
Jef Akst is a News Editor at The Scientist.