by Carl Zimmer
These experiments are now recognized as marking the birth of a synthetic biology. It was a humble start, when you consider that a clever child with a home electronics kit can make a blinking light or a toggle switch. But once biologists and engineers learn how to make simple genetic circuits, they move on to complex ones. Combine some simple logic gates and you can end up with a powerful computer chip.
I am writing this book only seven years after the birth of synthetic biology, and scientists are still a long way from building E. coli with the equivalent of a computer chip inside. But they have come a long way from toggle switches and blinkers. The E. coli camera is a good example of what they can do now. Each year Massachusetts Institute of Technology hosts a synthetic-biology tournament, in which students try to transform E. coli into various devices. In 2004, students at the University of Texas and the University of California, San Francisco, worked together to make bacteria that could capture an image. They envisioned a film of engineered E. coli that would behave like a piece of traditional photographic film. The bacteria would turn dark unless they were struck by light. The more light that struck them, the less dark they would become. Normally, E. coli cannot sense light, nor can it produce colors. But the students were able to engineer a strain that does both. They borrowed a gene for a light-sensitive receptor from a species of blue-green alga called Synechocystis. To color the microbes, they borrowed genes from Synechocystis that create pigments.
The hard part of the work came when it was time to join the two sets of genes. The students engineered the light receptors so that they could pass a signal to molecules normally made by E. coli. Those molecules were then able to grab on to the microbe’s DNA and shut down the production of Synechocystis’s pigment enzymes. It takes E. coli ten to fifteen hours of exposure to develop an image, which has a rather ghostly appearance. But because each microbe can adjust its own color, the photograph has a very high resolution, about ten times that of a high-resolution printer.
These sorts of experiments give synthetic biologists great hope. Soon it will be possible for them to synthesize entirely new genes from scratch at very little cost. No one can actually invent a completely new gene for a particular function, but it is possible to tinker with existing genes and simulate how their proteins would change as a result. Already researchers have fashioned new genes that allow E. coli to detect nerve gas and TNT. One of the most ambitious projects in all of synthetic biology is taking place at the University of California, Berkeley, where scientists have been developing new genetic circuits that may allow E. coli or yeast to produce a drug for malaria. The drug, known as artemisinin, is normally produced only by the sweet wormwood plant. If a microbe could make artemisinin, the price might drop by 90 percent.
Meanwhile, Christopher Voigt and his colleagues have created strains of E. coli that might someday fight cancer. The microbes seek out tumors by sensing their low levels of oxygen; having found a tumor, they deploy needles to inject toxins into the cancer cells. Voigt hopes someday to turn E. coli or some other microbe into a smart drug, able to make its own decisions about when to produce molecules to treat a disorder. Other researchers are trying to turn E. coli into a solar battery, able to trap sunlight and turn it into fuel. Synthetic biologists plan to move beyond E. coli, just as genetic engineers did. Someday they may be able to hack the programming of human cells, causing them to build new organs.
These are the things synthetic biologists think about when they’re in a good mood. When they’re in a bad mood, they think about all the challenges they still face.
Engineers, for example, need standardized parts. When engineers design a lathe or a lawn mower, they don’t have to design the nuts and bolts that hold the parts together. They just specify which size the nuts and bolts should be. Yet this shortcut is a relatively recent luxury. Before the mid-1800s, the threads on a nut made in one shop might not fit the threads on a bolt made in another. The standardization of those threads sped up the pace of invention and may even have played a major role in driving the Industrial Revolution.
For now, synthetic biology is a craft practiced by artisans. It took Elowitz and his colleagues—some of the world’s top experts on E. coli and its genes—more than a year to produce blinking bacteria. And once they had their successes, it was very difficult for other scientists to improve their circuits or incorporate them into more elaborate ones. For one thing, a scientist would have to reconstruct the circuit. And the circuit might work only in a particular strain of E. coli. Scientists can keep track of E. coli strains only with elaborate pedigree charts, tracing the bacteria like royalty. Such are the challenges that make engineers despair.
Since 2001, Drew Endy and Thomas Knight of MIT have been building a catalog of standardized parts for synthetic biology. If you want to add a toggle switch to your particular circuit, you can search for it on the BioBricks Web site, download the DNA sequence, order the corresponding fragments of DNA from a biotech firm, and insert them in E. coli. With more than 160 parts in its inventory, BioBricks has not only made synthetic biology easier but has also begun to foster a community. Endy and Knight made BioBricks the basis of the annual synthetic biology competition for students. The students themselves add more parts to the registry, opening the way for future inventions.
But as synthetic biologists try to build more ambitious circuits, they may find a new obstacle in their path: E. coli itself. For all of the attention scientists have lavished on it, there is still much about the microbe they do not understand. Six hundred genes remain absolute mysteries. The microbe’s genetic network is particularly murky. Scientists can identify most of E. coli’s transcription factors, the proteins that grab DNA to switch genes on and off, but they know only about half their targets. And what synthetic biologists do understand about E. coli sometimes makes their hearts sink. Its circuits overlap with one another, forming tangles that no self-respecting engineer would ever design. It is very hard to predict how extra circuits will change the behavior of such a messy network.
Some synthetic biologists are trying to overcome E. coli’s mystery by taking it apart and rebuilding it from scratch. At Harvard University, for example, George Church and his colleagues have drawn up a list of 151 genes, which they think would be enough to keep an organism alive. Scientists understand these genes—which are drawn mostly from E. coli and its viruses—quite well. There should be relatively little mystery when they come together. Church hopes to create a genome with these essential genes. By combining it with a membrane and protein-building ribosomes, he hopes to create a living thing. Call it E. coli 2.0.
Meanwhile, at Rockefeller University, Albert Libchaber took an even simpler approach. He and his colleagues cooked up a solution of ribosomes and other molecules found in E. coli. Instead of a full genome, they engineered a small plasmid. They then added oily molecules from egg yolks, which form bubbles that scoop up the genes and molecules. These bubbles, Libchaber’s team found, could live—at least for a few hours. One of the genes Libchaber added to the plasmids encoded a pore protein. The protocells read the gene, built the proteins, and inserted them in the membrane. There they could allow amino acids and other small molecules to move into the protocell without letting the plasmid and other big molecules out. To track the production of new proteins, the scientists also added a gene from a firefly. The protocells gave off a cool green glow. Libchaber doesn’t call his creation a living thing. He prefers the term bioreactor. To go from bioreactor to life will take much more work. For one thing, Libchaber and his colleagues will need to add genes to allow the bioreactors to divide into new bioreactors.
Church and Libchaber are only just starting to figure out how to use parts of E. coli to create new life-forms. They cannot just throw together DNA and some other molecules and let them come to life on their own. Life is not like a computer, which simply boots up at the press of a button. Every E. coli alive today emerged from an ancestor, which emerged from ancestors of its own. Together they form an unbroke
n river of biology that has flowed continuously for billions of years. Life as we know it has always been part of that river. Perhaps now we will make a canal of our own.
RETURN OF FRANKENSTEIN’S MICROBE
In May 2006, synthetic biologists met in Berkeley, California, for their second international meeting. Along with the standard research talks, they set aside time to draft a code of conduct. The day before, thirty-five organizations—representing, among others, environmentalists, social activists, and biological warfare experts—released an open letter urging that the biologists withdraw the code. They should join a public debate about synthetic biology instead and be ready to submit to government regulations. “Biotech has already ignited worldwide protests, but synthetic biology is like genetic engineering on steroids,” said Doreen Stabinsky of Greenpeace International.
These days, biotechnology is experiencing an intense case of déjà vu. The questions people are debating about synthetic biology are strikingly similar to the ones that came up when genetically engineered E. coli made news in the 1970s. Do the benefits justify the risks? Is there any intrinsic wrong in tinkering with life? The new debate is far more complex than the old one, in part because E. coli is not the only thing scientists are manipulating. Now we must consider transgenic crops, engineered stem cells, human-animal chimeras. The new debate often turns on subtle points of medicine, conservation biology, patent law, and international trade. But for all the differences, the parallels are still powerful and instructive. To understand the potential risks and benefits of the new biotechnology, it helps to look back at the fate of genetically engineered E. coli over the past three decades.
The dire warnings that E. coli would create tumor plagues and insulin shock epidemics seem quaint today. In thirty years no documented harm from genetically engineered E. coli has emerged, despite the fact that many factories breed the bacteria in 40,000-liter fermenters in which every milliliter contains a billion E. coli. No one has a God’s-eye view of the fate of every engineered E. coli in the past thirty years, so it’s impossible to know for sure why the predicted plagues never came. Some clues have come from experiments. Scientists put E. coli K-12 carrying human genes in tubs of sludge and tanks of water and animal guts. They found that the bacteria rapidly disappeared. Genetically engineered E. coli channel a lot of energy and raw materials into making the proteins from inserted genes. But those proteins, such as insulin and blood thinners, probably don’t boost E. coli’s growth or odds of surviving in the wild. In the carefully controlled conditions scientists create in laboratories, they can thrive. But pitted against other bacteria, they fail.
Genetic engineers did not introduce genes to E. coli from other species for the first time. In a sense, E. coli and its ancestors have been genetically engineered for billions of years. But most of the transfers have been complete failures. Bacteria cannot make proteins from many horizontally transferred genes, and natural selection favors mutations that strip most alien genes from their genomes.
Unfortunately, the absence of evidence is not a slam-dunk case for the evidence of absence. If an engineered strain of E. coli escapes from a factory and manages to survive in the outside world for a few days, it may be able to pass its genes to other bacteria. If a soil microbe picks up a gene for human insulin or some other alien protein, it probably would not benefit from it. But the possibility can’t be ruled out. Studies suggest that even if an alien gene gave bacteria a competitive advantage, it would remain too rare for scientists to detect for decades, perhaps even centuries.
While we’ve been waiting for a genetically engineered monster to emerge, E. coli O157:H7 has emerged as a serious threat to public health. It was in 1975—the same year in which scientists gathered at Asilomar to ponder the potential dangers of genetically engineered E. coli—that a woman suffered the earliest known attack of E. coli O157:H7. But that pathogen was not the work of a human genetic engineer with an intelligent design. Over the course of centuries, E. coli O157:H7 acquired many genes from viruses carrying deadly instructions. They acquired these genes from other strains of E. coli or other species of bacteria. They acquired syringes and toxins and molecules that alter the behavior of host cells. This genetic engineering is still taking place as one new strain after another evolves. But the insertion of a bundle of genes in a single microbe was only the first step in this transformation. Natural selection then had to favor those genes in their new host; it had to fine-tune them.
The transformation required an entire ecosystem that could produce the conditions that would drive natural selection. We provided it. E. coli O157:H7 had been pumped from humans to livestock through farm fields and slaughterhouses, through rivers and sewers rife with toxin-bearing viruses. There’s little evidence for a similar evolutionary pump for genetically engineered E. coli. Our unplanned engineering of E. coli may give us more to worry about than anything brewed up in a lab.
Thirty years have passed since the backers of genetic engineering predicted recombinant DNA would bring great rewards. They were right, up to a point. E. coli and other engineered cells not only produce a vast number of valuable molecules; they have also sped up the pace of science enormously. E. coli was a crucial partner in the sequencing of the human genome, for example. In order to read the genome, scientists inserted chunks of it into E. coli, which then produced many copies that scientists could analyze. Other scientists have used E. coli to churn out millions of proteins so that they can discover what the proteins do. By inserting human genes into E. coli, scientists discovered that they are made up of two kinds of DNA. Some segments of the genes, known as exons, encode parts of proteins. But they alternate with other segments, called introns, that encode nothing. Our cells edit out the introns from RNA in order to make proteins. They can even use different combinations of exons to produce a number of proteins from a single gene.
As important as these accomplishments have been, however, genetic engineering has fallen far short of the more extravagant promises offered thirty years ago. Cetus predicted that all major diseases would surrender to genetically engineered proteins by 2000. I’m writing in 2007, and cancer, heart disease, malaria, and other diseases continue to kill by the millions. Maybe the people at Cetus were just wrong about the date. Perhaps another thirty years will bring some major breakthrough in genetic engineering that will wipe out all major diseases. I wouldn’t bet on it, though. Most major diseases are fiendishly complex, and a single engineered protein is not going to make them go away. Diabetes, the poster child for the promise of genetic engineering, has not disappeared over the past thirty years. In fact, it has exploded. The incidence of type 2 diabetes has doubled in the United States, and cases of diabetes worldwide have increased tenfold. E. coli has provided insulin for millions of people with diabetes, but, as Ruth Hubbard warned, it did nothing to prevent the disease. Genetic engineering could not block the sources of the diabetes epidemic, which may include the availability of cheap sugar. That sugar comes increasingly from high-fructose corn syrup, whose low price we owe to breakthroughs in genetic engineering.
Drugs made through genetic engineering have also turned out to be just as vulnerable to market forces as conventional ones. Drug companies have been trying to increase their sales by expanding our definition of what it means to be sick. Genetically engineered drugs have been promoted this way as well. Genentech originally got approval from the Food and Drug Administration to sell its E. coli–produced growth hormone to treat children whose bodies couldn’t make it themselves. But in 1999 the company had to pay $50 million to settle charges that its drug was being marketed to children who were merely shorter than average.
E. coli’s thirty-year history of genetic engineering is worth considering when we judge the new biotechnology that has come in its wake. We must resist empty fear and empty hype. We must instead be realistic, always remembering how both nature and society actually work.
One of the great dreams of biotechnology has been to end famine, for example. Julian
Huxley speculated as far back as 1923 that scientists would create a limitless supply of food (along with purple oceans). The dream lived on in the 1960s with promises of oil-fed yeast. When scientists successfully inserted foreign genes in E. coli, advocates for genetic engineering promised more food for a starving world. In the 1970s, the Green Revolution—the result of breeding new varieties of crops and using plenty of fertilizer—had dramatically increased farm productivity. But the world’s population, and thus its hunger, were still growing. Scientists began trying to engineer bacteria to make fertilizer by capturing nitrogen from the air. Most recently, scientists have turned their attention to engineering plants themselves. Transgenic crops are being promoted not as a way to make bigger profits but as a way to fight hunger and malnutrition. Crops that can resist viruses and insects will increase harvests. Crops that can resist herbicides will allow farmers to fight weeds more effectively, increasing the yield even more. Norman Borlaug, who won a Nobel Peace Prize for his work on the Green Revolution, claimed that genetically modified crops would pick up where his own work had left off, feeding the world for another century.
Anyone who questioned this prediction, Borlaug suggested, was dooming the world’s poor to famine. “The affluent nations can afford to adopt elitist positions and pay more for food produced by the so-called natural methods; the 1 billion chronically poor and hungry people of this world cannot,” he wrote in 2000. “New technology will be their salvation, freeing them from obsolete, low-yielding, and more costly production technology.”
One of the promising crops Borlaug—as well as many other advocates—pointed to was Golden Rice, a strain of rice engineered to make vitamin A. Vitamin A deficiency affects roughly 200 million people worldwide. Up to half a million children become blind each year, half of whom will die within a year of losing their sight. In the late 1990s, Swiss scientists began inserting genes from daffodils and bacteria into the rice genome to produce vitamin A. They formed a partnership with the corporation Syngenta to develop the rice and distribute it free to farmers who make less than $10,000 a year. Ingo Potrykus, one of the inventors, appeared on the cover of Time in 2000, alongside the headline “THIS RICE COULD SAVE A MILLION KIDS A YEAR,” which was followed in small print by “…but protesters believe such genetically modified foods are bad for us and our planet. Here’s why.”
