by Carl Zimmer
Instead of a miracle, critics saw in genetic engineering the illusion of a quick fix. In 1977, the National Academy of Sciences held a public forum on the risks and benefits of the new technology. Picketers tried to shut down the meeting, calling genetic engineers Nazis. Amid the chaos, Irving Johnson, the vice president of research at Eli Lilly, talked about how genetic engineering could be used to treat diabetes. Eli Lilly, the country’s biggest provider of insulin, got the hormone from the pancreases of pigs. That supply was vulnerable, Johnson said, to a slump in the pork business or to an increase in the population of diabetics. Genetically engineering a microbe to make human insulin might provide a vast, cheap supply. “This is truly ‘science for the people,’” Johnson said.
Ruth Hubbard, a Harvard biologist and a leading critic of genetic engineering, testified against this sunny view. She pointed out that insulin does not prevent diabetes or even cure it. It merely counteracts some of the symptoms of the disease. “Before we jump at technological gimmicks to cure complicated diseases,” she warned, “we first have to know what causes the diseases, we have to know how the therapy that we are being told is needed works, we have to know what fraction of people really need it…. But what we don’t need right now is a new, potentially hazardous technology for producing insulin that will profit only the people who are producing it.”
While genetic engineering was distracting society from real solutions, critics warned, it could also put the world at risk. What made it particularly risky was its utter dependence on E. coli. “From the point of public health,” Cavalieri declared, “this bacterium is the worst of all possible choices. It is a normal inhabitant of the human digestive tract and can easily enter the body through the mouth or nose. Once there, it can multiply and remain permanently. Thus every laboratory working with E. coli recombinants is staffed by potential carriers who could spread a dangerous recombinant to the rest of the world.”
Even if scientists used a weakened strain of E. coli for genetic engineering, the microbes might survive long enough outside a lab to pass their engineered genes to more rugged strains. Critics warned of cancer epidemics caused by E. coli casually poured down a drain. E. coli might churn out insulin inside diabetics, sending them into comas. Genetically engineered organisms could cause bigger disasters than toxic chemicals because they had the reproductive power of life. Erwin Chargaff, an eminent Columbia University biologist, called genetic engineering “an irreversible attack on the biosphere.”
“The world is given to us on loan,” Chargaff warned. “We come and we go; and after a time we leave earth and air and water to others who come after us. My generation, or perhaps the one preceding mine, has been the first to engage, under the leadership of the exact sciences, in a destructive colonial warfare against nature. The future will curse us for it.”
These attacks left the champions of genetic engineering stunned. The debate had become “nightmarish and disastrous,” Paul Berg declared in 1979. Stanley Cohen called it a “breeding ground for a horde of publicists.”
James Watson, as usual, was bluntest of all. “We were jackasses,” he said, looking back at his support of the 1974 moratorium. “It was a decision I regret; one that I am intellectually ashamed of.” It had led the public to distract itself from real threats with illusions of apocalypse.
“I’m afraid that by crying wolf about dangers which we have no reason at all to worry about, we are becoming indistinguishable from my two small boys,” he wrote. “They love to talk about monsters because they know they will never meet one.”
E. COLI, INC.
One figure noticeably absent from the debate was Herbert Boyer, the scientist who had triggered the genetic engineering controversy in the first place. He was busy hunting for companies and investors who could help him make money from his restriction enzymes. In 1976, he became a partner with a young entrepreneur named Robert Swanson. Each man ponied up $500 to launch a company they called Genentech (short for genetic engineering technology). Boyer had to borrow his share.
Boyer and Swanson set out to sell valuable molecules produced by engineered E. coli. They decided their first goal should be human insulin, for many of the reasons Irving Johnson had offered to the National Academy of Sciences. Boyer turned to Arthur Riggs and Keiichi Itakura at the City of Hope Hospital in Duarte, California, for help. Riggs and Itakura were among the first scientists learning how to build genes from scratch. When Boyer contacted them, they were in the midst of synthesizing their first human gene, which encoded the hormone somatostatin. Working with Genentech, Riggs and Itakura figured out how to add sticky ends to an artificial somatostatin gene and insert it into a plasmid. They put the plasmid in E. coli, which then began to produce somatostatin. It was yet another milestone in a very young science. In 1973, Boyer, Cohen, and Morrow had managed only to put a fragment of an animal gene in E. coli. Four years later, Genentech had E. coli that could make human proteins.
The scientists did not take long to savor the glory. After they announced their results in 1977, they moved on to insulin. Boyer knew he would have to move fast. Walter Gilbert, the brilliant Harvard molecular biologist, was trying to make insulin as well. But Boyer had a crucial advantage over Gilbert: Boyer’s DNA was artificial. Gilbert was trying to isolate insulin DNA from real cells, so his research was subject to the tight grip of government regulation. His team had to take extraordinary precautions and even flew to England to work in a lab set up for biological warfare research. Boyer could move faster because his DNA was not “natural.” Instead of isolating it from a cell as Gilbert was doing, Riggs and Itakura worked their way backward from the insulin protein to the sequence of the insulin’s gene. Free of regulations, Boyer won the race. On September 6, 1978, Genentech announced that its scientists had extracted 20 billionths of a gram of human insulin from E. coli.
Over the next two years, Genentech researchers boosted the yield. They engineered E. coli so that it would push its insulin out of its membrane, making it easier to harvest. In 1980, Genentech was ready to hand over the production of insulin to Eli Lilly. The following year the pharmaceutical giant built 40,000-liter tanks, in which it began to breed E. coli. Genentech went public, and Boyer’s $500 became $66 million.
As Genentech’s fortunes waxed, the controversy over E. coli waned. Congress never passed a genetic engineering bill, thanks in part to fierce lobbying by scientists. The National Institutes of Health relaxed its guidelines. Scientists working on E. coli no longer had to dress up in space suits. Corporations snatched up E. coli experts in increasing numbers. All fourteen signatories to Paul Berg’s original moratorium letter ended up associated with one venture or another. Walter Gilbert helped launch a company called Biogen, which began engineering E. coli to spew out proteins that showed promise of fighting cancer. When Biogen opened its headquarters in Cambridge, Gilbert’s old nemesis, former mayor Alfred Vellucci, was there to cut the ribbon.
Genentech led the way for the new biotechnology. Humulin, its microbe-produced insulin, went on the market in 1983, and now 4 million people worldwide take the drug. Other companies make their own brands of E. coli–produced insulin, which are used by millions of other diabetics. Biotechnology firms have developed many other drugs from E. coli, ranging from human growth hormone to blood thinners. Today E. coli churns out vitamins and amino acids. Traditionally, cheese is made by spiking milk with rennet, an enzyme produced in cows’ stomachs. Now much of the cheese in stores is made with rennet produced by E. coli. Scientists are adding new genes to E. coli to see what sorts of new things they can produce, from biodegradable plastics to gasoline.
These advances have not come easily. Scientists cannot simply treat E. coli as a machine. The microbe is a living thing, and it responds to manipulation in unexpected ways. Packing the bacteria in a giant tank can cause them to suffocate in their own waste. Engineering them to produce huge amounts of insulin or some other foreign protein puts them under tremendous stress. If the proteins clump together,
E. coli produces heat-shock proteins to try to untangle them. All the energy E. coli uses up coping with the stress is energy it cannot use to feed and grow. Scientists, like cooks perfecting recipes, have struggled to find solutions to these quandaries.
Thirty years have now passed since E. coli became the monster and the mule of genetic engineering. It remains one of biotechnology’s favorite microbes. Scientists continue to experiment on it to find new ways to manipulate genes and proteins. Its restriction enzymes are the blade of choice for slicing DNA, and its plasmids are the favored breeders of new copies of genes. But scientists can now insert these genes in many other species as well. In the 1980s, they began using the lessons they learned from E. coli to shuttle genes into other bacteria and into fungi. Scientists have also learned how to introduce genes into animal and plant cells. Paul Berg’s original dream has become real: it is now possible to load a gene on a virus such as SV40 and infect an isolated mammal cell. (Cells from the ovaries of Chinese hamsters are a popular choice.) An engineered cell can then multiply into a laboratory colony, which can then churn out a valuable protein.
It’s now also possible to inject genes into living plants. Genetically modified crops now grow across vast stretches of farmland in many countries. Some crops produce a toxin normally made by bacteria that kills insects. Others have been engineered to withstand a weed killer. Scientists have also succeeded in creating plants that can produce human antibodies and vaccines.
Even animals now acquire foreign genes from engineered viruses. Some researchers hope they will be able to treat genetic disorders by supplying cells with working copies of essential genes. Others are inserting genes directly into embryonic cells to produce animals with foreign genes throughout their bodies. Some scientists are trying to ease the pollution produced by farms with this sort of genetic engineering. One major form of pollution from farms is the phosphates—compounds of phosphorus and oxygen—concentrated in fertilizer. When fertilizer washes out into rivers and oceans, the phosphates cause algae blooms and other ecological upheavals that eventually create vast dead zones where nothing can survive. One reason for the high levels of phosphates in fertilizer is that much of it comes from the manure of livestock such as pigs and chickens. These animals cannot break down the phosphates in their food, so it just goes straight through their digestive systems. E. coli, among other bacteria, make enzymes that can break down those phosphate-bearing molecules. When researchers insert E. coli’s genes in pigs, the animals produce manure that has only a quarter of the normal level of phosphates.
It’s chimeric turnaround: thirty years ago scientists were putting animal genes into E. coli. Now they are giving animals the genes of E. coli.
EXPANDING LIFE’S ALPHABET
Herbert Boyer used his intimate knowledge of E. coli’s biology to help create genetic engineering. Today scientists are using his tools to learn more about E. coli itself. In the process, they’re answering some of the most fundamental questions about life.
Scientists have long debated why life on Earth, with almost no exception, uses only twenty amino acids to build proteins. (E. coli is unusual in its ability to make a twenty-first amino acid, called selenocysteine.) There are hundreds of perfectly respectable kinds of amino acids life could have chosen from. To join the Amino Acid Club, a molecule needs only the proper ends. It must have a cluster of nitrogen and hydrogen atoms at one end (an amine) and a cluster of carbon, hydrogen, and oxygen on the other (a carboxyl group). An amine from one amino acid snaps onto the carboxyl group of another like LEGO pieces. It matters little what lies in between. A chemist can synthesize hundreds of different amino acids, and so can the chemistry of outer space. In 1969, a meteorite coated with tarry goo fell to Earth. Scientists found seventy-nine kinds of amino acids lurking inside it.
So why do we have just twenty? One way to investigate the question is to try to produce an organism that can make more. In 2001, Peter G. Schultz of Scripps Research Institute in La Jolla, California, and his colleagues did just that, by engineering E. coli. Like other living things, E. coli uses a genetic code in which three bases of DNA translate into one amino acid. There are sixty-four possible codons in E. coli’s genetic code, most of which it uses regularly. Schultz and his colleagues identified one that it uses only rarely. They engineered E. coli so that this neglected codon now instructed the microbe to add an unnatural amino acid to a protein.
Science magazine hailed the achievement as “the first synthetic life form with a chemistry unlike anything found in nature.” In the years since, scientists have added over thirty more unnatural acids to E. coli’s repertoire. Originally E. coli could make these new proteins only if it was supplied with the unnatural amino acids. Recently scientists have begun engineering E. coli to make unnatural amino acids from its natural food.
This research has pushed the debate over the genetic code to new ground. No one can argue that life’s twenty amino acids are the only ones that can make life possible. Some scientists now argue that the genetic code is just a historical artifact. Early life built its proteins with the most abundant amino acids on the planet, and that unconscious choice was frozen in place. Other scientists argue that the genetic code is actually the best of all possible codes. It offers the biggest range of potential proteins with the fewest genes. And still other scientists argue that natural selection produced the genetic code because it is robust, with the least risk of producing a lethally deformed protein if a mutation strikes a gene.
In our hands, however, the rules of the genetic code have changed. Schultz and other researchers are looking for practical applications for E. coli’s unnatural proteins. Unnatural proteins may allow E. coli to overcome one of genetic engineering’s biggest failures. Unlike bacteria, human cells decorate many of their proteins with knobs of sugar. The sugars force the proteins into new shapes, allowing them to take on new functions. E. coli can make perfect copies of our proteins, amino acid for amino acid, but if it can’t add the sugars, many of its proteins are useless to us.
Schultz and his colleagues have found a way around this shortcoming. Instead of adding the sugar after a protein is built, they add it to individual amino acids. They then engineer E. coli to recognize the unnatural sugarcoated amino acids instead of the ones it normally uses. In this arrangement the bacteria can assemble proteins with sugar knobs already in place, ready for human consumption. What is unnatural for E. coli turns out to be quite natural for us.
NETWORK HACKS
For all the futuristic aura around genetic engineering, the science is rather quaint. It is based on a 1950s view of biology. In the world of genetic engineering, E. coli and other species are nothing more than simple chemical factories manufacturing their own sets of proteins. Change a gene and you change one of the proteins that comes out. Genetic engineers are well aware that there is much more to life than the production of proteins. There are repressors and promoters, for example, which turn genes on and off. But many genetic engineers use these insights only to make E. coli and other organisms into even better factories.
There’s another way to look at E. coli: as a network. Its proteins and genes work together, allowing the microbe to process information, to make decisions, to keep its biology steady in an unsteady world. The powers of this network emerge from the sum of its parts, not from any one gene or protein. Engineers regularly improve on man-made networks—rewiring circuits, swapping parts. If life follows engineering principles as well, some scientists wonder, would it be possible to rewire life, too?
The first two reports of rewired life came in 2000, and in both cases the life in question was E. coli. Michael Elowitz at California Institute of Technology and Stanislas Leibler of Rockefeller University in New York made the microbe blink. They used three genes to build a circuit. Each gene made a different repressor. Elowitz and Leibler engineered the first gene so that its repressor shut down the second gene. The repressor made from the second gene shut down the third. The third shut down the first, b
ut it also did something else: it caused E. coli to build a glowing-jellyfish protein.
Elowitz and Leibler found that in some of their engineered microbes, the three repressors became locked in a cycle. As the first gene made more and more repressors, it shut down the activity of the second gene, freeing the third gene to shut down the first one. As the first one stopped making its repressor, the second gene was freed and shut down the third gene, and so on. Elowitz and Leibler arranged these genes on a plasmid and inserted them in E. coli. As the genes became active, the scientists could witness this cycle with their own eyes: as the third gene switched on and off, it produced more and then less light. In other words, E. coli blinked.
The second report came from the laboratory of James Collins at Boston University. Collins and his colleagues gave E. coli a toggle switch. They built two genes, each encoding a repressor that shut off the other gene. Each repressor could be pulled off E. coli’s DNA by adding a different molecule to the microbe. To observe how this new circuit of genes worked, Collins and his colleagues, like Elowitz and Leibler, added instructions to one of the genes for building a glowing protein. Adding one kind of molecule caused E. coli to start glowing and to continue glowing even after the molecule had run out. Adding the other kind of molecule shut the glow down and kept E. coli dark even after it, too, had run out.
