by W. Wayt Gibbs
April 26, 2004
from GeneticEngineeringAndItsDangers Website
of interchangeable DNA parts
and assembling them inside microbes
to create programmable, living machines
Evolution is a wellspring of creativity; 3.6 billion years of mutation and competition have endowed living things with an impressive range of useful skills.
But there is still plenty of room for improvement. Certain microbes can digest the explosive and carcinogenic chemical TNT, for example - but wouldn't it be handy if they glowed as they did so, highlighting the location of buried land mines or contaminated soil?
Wormwood shrubs generate a potent medicine against malaria but only in trace quantities that are expensive to extract.
How many millions of lives could be saved if the compound, artemisinin, could instead be synthesized cheaply by vats of bacteria? And although many cancer researchers would trade their eyeteeth for a cell with a built-in, easy-to-read counter that ticks over reliably each time it divides, nature apparently has not deemed such a thing fit enough to survive in the wild.
Biologists have been transplanting genes from one species to another for 30 years, yet genetic engineering is still more of a craft than a mature engineering discipline.
but as they do, they mutate.
Endy is one of a small but rapidly growing number of scientists who have set out in recent years to buttress the foundation of genetic engineering with what they call synthetic biology.
They are designing and building living
systems that behave in predictable ways, that use interchangeable
parts, and in some cases that operate with an expanded genetic code,
which allows them to do things that no natural organism can.
The current prototypes are relatively
primitive, but the vision is undeniably grand: think of it as Life,
In 1989 Benner led a team at ETH Zurich that created DNA containing two artificial genetic "letters" in addition to the four that appear in life as we know it. He and others have since invented several varieties of artificially enhanced DNA. So far no one has made genes from altered DNA that are functional - transcribed to RNA and then translated to protein form - within living cells.
Just within the past year, however,
Schultz's group at the Scripps Research Institute developed cells
(containing normal DNA) that generate unnatural amino acids and
string them together to make novel proteins.
Two such devices, reported
simultaneously in 2000, inspired much of the work that has happened
Meanwhile James J. Collins,
Charles R. Cantor and Timothy S. Gardner of Boston
University made a genetic toggle switch. A negative feedback loop -
two genes that interfere with each other - allows the toggle circuit
to flip between two stable states. It effectively endows each
modified bacterium with a rudimentary digital memory.
And no one could see a way to connect the two devices to make, for example, blinking bacteria that could be switched on and off.
Four years ago parts such as these were just a dream.
Today they fill a box on Endy's desk.
At a mechanical level, individual BioBricks (as the M.I.T. group calls the parts) can be fabricated and stored separately, then later stitched together to form larger bits of DNA. And on a functional level, each part sends and receives standard biochemical signals.
So a scientist can change the behavior of an assembly just by substituting a different part at a given spot.
One advantage it offers is abstraction.
Just as electrical engineers need not know what is inside a capacitor before they use it in a circuit, biological engineers would like to be able to use a genetic toggle switch while remaining blissfully ignorant of the binding coefficients and biochemical makeup of the promoters, repressors, activators, inducers and other genetic elements that make the switch work.
One of the vials in Endy's box, for example, contains an inverter BioBrick (also called a NOT operator). When its input signal is high, its output signal is low, and vice versa.
Another BioBrick performs a
function, emitting an output signal only when it receives high
levels of both its inputs. Because the two parts work with
compatible signals, connecting them creates a
NAND (NOT AND)
operator. Virtually any binary computation can be performed with
enough NAND operators.
The students did not know how to create DNA sequences, but they had no need to. Endy hired a DNA-synthesis company to manufacture the 58 parts called for in their designs. These new BioBricks were then added to M.I.T.'s Registry of Standard Biological Parts.
That online database today lists more
than 140 parts, with the number growing by the month.
Electrical and mechanical machines are generally self-contained. That is true for a select few genetic devices: earlier this year, for example, Milan Stojanovic of Columbia University contrived test tubes of DNA-like biomolecules that play a chemical version of tic-tac-toe.
But synthetic biologists are mainly interested in building genetic devices within living cells, so that the systems can move, reproduce and interact with the real world. From a cell's point of view, the synthetic device inside it is a parasite.
The cell provides it with energy, raw
materials and the biochemical infrastructure that decodes DNA to
messenger RNA and then to protein.
And yet, acknowledges Ron Weiss of Princeton,
Indeed, Endy recalls,
One way to deal with the complexity added by the cells' native genome is to dodge it: the genetic device can be sequestered on its own loop of DNA, separate from the chromosome of the organism. Physical separation is only half the solution, however, because there are no wires in cells.
Life runs on "wetware," with many protein signals simply floating randomly from one part to another.
One way to prevent crossed signals is to avoid using the same part twice.
Weiss has taken this approach in
constructing a "Goldilocks" genetic circuit, one that lights up when
a target chemical is present but only when the concentration is not
too high and not too low. Tucked inside its various parts are four
inverters, each of which responds to a different protein signal. But
this strategy makes it much more difficult to design parts that are
truly interchangeable and can be rearranged.
In principle, the inverter could be
removed and replaced with any other BioBrick that processes
And TIPS signals are location-specific, so the same part can be used
at several places in a circuit without interference.
To do this the cells must communicate with one another by secreting and sensing chemical nutrients.
It took 13 months to get the blinking E. coli designs built and into cells.
But in the intervening year the inventory of BioBricks has grown, the speed of DNA synthesis has shot up, and the engineers have gained experience assembling genetic circuits.
So Endy expects to have the 2004 designs ready for testing in just five months, in time to show off at the first synthetic biology conference, scheduled for this June.
Living machines reproduce, but as they do they mutate.
Weiss and Frances H. Arnold of the California Institute of Technology have evolved circuits with improved performance using multiple rounds of mutation followed by selection of those cells most fit for the desired task.
But left unsupervised, evolution will tend to break genetic machines.
Or perhaps the engineers will have to understand better how simple forms of life, such as viruses, have solved the problem of persistence. Synthetic biology may help here, too.
Last November, Hamilton O. Smith and J. Craig Venter announced that their group at the Institute for Biological Energy Alternatives had re-created a bacteriophage (a virus that infects bacteria) called phiX174 from scratch, in just two weeks.
The synthetic virus, Venter said, has the same 5,386 base pairs of DNA as the natural form and is just as active.
Re-creating a virus letter-for-letter does not reveal much about it, but what if the genome were dissected into its constituent genes and then methodically put back together in a way that makes sense to human engineers?
That is what Endy and colleagues are doing with the T7 bacteriophage.
The scientists are separating genes that overlap, editing out redundancies, and so on.
The group has completed about 11.5
kilobases so far and expects to finish the remaining 30,000 base
pairs by the end of 2004.
But a number of research laboratories are already working on applications. Martin Fussenegger and his colleagues at ETH Zurich have graduated from bacteria to mammals. Last year they infused hamster cells with networks of genes that have a kind of volume control: adding small amounts of various antibiotics turned the output of the synthetic genes to low, medium or high.
Controlling gene expression in this way
could prove quite handy for gene therapies and the manufacture of
Weiss says that he and Hellinga have
discussed combining his Goldilocks circuit with Hellinga's sensor to
make land-mine detectors.
The circuit enables the bacterium to
fabricate a chemical precursor to artemisinin, a next-generation
antimalarial drug that is currently too expensive for the parts of
the developing world that need it most.
By boosting the yields another 25- to 50-fold, he adds,
With relatively simple modifications,
the bioengineered bacteria could be altered to produce expensive
chemicals used in perfumes, flavorings and the cancer drug
One team is modifying the bacteria's sense of "smell" so that the bugs will swim toward a nerve agent, such as VX, and digest it.
Worthy goals, all.
But if you become a
touch uneasy at the thought of undergraduates creating new kinds of
germs, of private labs synthesizing viruses, and of scientists
publishing papers on how to use bacteria to collect plutonium, you
are not alone.
Self-policing seemed to work: there has yet to be a major accident with genetically engineered organisms.
So how does society counter the risks of a new technology without also denying itself all the benefits?
He pulls out a photograph of the class he taught last year.
But he also believes that a meeting to address potential problems makes sense.
This June, as leaders in the field meet
to share their latest ideas about what can now be created, perhaps
they will also devote some thought to what shouldn't.