Evolution continuously shapes life on our planet, all the way down to an organism’s molecular machinery. Surviving genetic variants thrive and move to the next round, where mutations might further improve them. The less fit perish.
This kind of natural selection can be a gradual and drawn-out process. But HHMI investigator David Liu wants speed.
Liu and his team have developed a system that uses phage, the viruses that infect bacteria, to accelerate evolution in the laboratory. By tying a virus’s fate to the function of a specific protein or nucleic acid, the system can improve that molecule’s existing ability, or coax it to evolve a new one. Called phage-assisted continuous evolution, or PACE, the system ushers the evolution of molecules along approximately 100 times faster than previous techniques.
It offers an exciting prospect for developing new pharmaceuticals as well as studying evolution.
A typical single round of directed evolution takes days to complete, and many rounds may be necessary to achieve a researcher’s goals. It’s tedious work, and a gamble. Even after dozens of evolutionary rounds—more than researchers can typically handle—targeted improvements to a molecule may be modest. Scientists patient enough to spend years with the process also bemoan the need to babysit equipment, reagents, microbes, and genetic sequences along the way.
Liu, a chemical biologist at Harvard University, and his graduate students Kevin Esvelt and Jacob Carlson wanted an end to the monotony. Their PACE system replaces the human grunt work of laboratory evolution with a partnership between fast-reproducing M13 bacteriophage and Escherichia coli bacteria. The system is a veritable self-sustained factory of biomolecular evolution.
In their first report describing PACE, published April 28, 2011, in Nature, Liu and his team used the system to continuously evolve an enzyme to gain three new abilities. In each case, the new version of the enzyme showed activity levels as high as, or higher than, the naturally occurring enzyme. In one example, they put a protein through 200 rounds of evolution in only eight days.
“Using a conventional protein evolution method, it would have taken us years,” Liu says. “The speed and throughput of PACE is pretty hard to match.”
In the system, a never-ending current of fluids and E. coli bacteria creates a life-or-death pressure for M13 bacteriophage. To survive in this evolutionary “river,” the phage must in effect swim upstream by infecting E. coli and reproducing more quickly than they are washed away. Since a phage can churn out hundreds of copies of itself in 10 minutes, this feat should not be a problem.
To harness phage to perform evolution, however, Liu can’t use your garden variety phage. Each phage contains the gene for the protein the researcher wants to evolve, but the phage genome is missing a critical element: gene III, a protein-encoding gene necessary for progeny phage to infect E. coli. The researchers hoped to force the phage to evolve proteins of interest by linking the proteins’ desired activities to the activation of gene III expression. Indeed, making gene III expression dependent on the desired activity of the enzyme resulted in the breathtaking evolution of their target protein.
For the target protein in their Nature study, Liu and his team chose T7 RNA polymerase, an enzyme that transcribes T7 bacteriophage DNA into RNA. Beginning with a phage encoding the wild-type gene for T7 RNA polymerase, the team hoped to rapidly evolve three different transcription-related activities in the enzyme as a proof of concept.
The gene III construct was spliced into a plasmid—a small, customized genome—and inserted into host E. coli cells. Only phage with a T7 RNA polymerase gene capable of one of the three target activities could turn on gene III in the plasmid. To survive, a phage had to contain a functional copy of T7 RNA polymerase. Any phage with nonfunctional copies would flow into the waste bin.
“Very rapidly, in hours to days, we ended up with highly mutated genes of interest that had dramatic increases in desired activity levels,” Liu says. One PACE-evolved enzyme, for example, showed a several hundredfold increase in its ability to recognize a new promoter and begin transcribing it into RNA.
To boost the rate further, Liu’s team slipped a second plasmid into the bacteria that spiked mutation rates to levels 100 times higher than normal (see diagram). The trick helped PACE spawn billions of mutated T7 RNA polymerase genes every 10 to 20 minutes.
Liu points out that PACE can’t be used to evolve every protein or nucleic acid activity—only those that can be tied directly or indirectly to expression are PACE compatible. But he anticipates that the system will find broad use to evolve many proteins, including polymerases, protein-cleaving enzymes, protein–protein binding partners, and those that regulate genes.
“Beyond functional applications, PACE can also be used to answer fundamental questions that require hundreds or thousands of rounds of evolution,” Liu says. “For example, if you repeat 1,000 rounds of evolution 10 times under identical conditions, replaying ‘the tape of life’ as Stephen Jay Gould called it, do you get different outcomes or similar outcomes? And what affects those outcomes?
“This is the first time we’ve had practical means to seek answers to questions like this over long evolutionary trajectories,” he says.