research highlights

molecular engineering Directed evolution made easy

Phage-assisted continuous evolution of reproduce and infect incoming bacteria. proteins automates and accelerates selec- The better the phage is at inducing gene tion, allowing hundreds of rounds of evo- expression in the accessory plasmid, the lution to occur in a single week. more phage are produced. Rounds of selec- Researchers who want to engineer a pro- tion continue as long as fresh E. coli are fed tein to bind tightly to an antigen or to cata- into the lagoon, but ‘successful’ phage can lyze a given reaction often use a strategy be isolated, sequenced and characterized at called directed evolution. This approach any point. randomly generates many variants of a To see whether the process could evolve gene of interest, then selects those versions proteins with desired properties, the encoding proteins that are best at per- researchers set out three tests. Starting with forming some desired activity. In a typical the same T7 RNA polymerase, researchers round of evolution, genetic variants are selected for new versions that recognized a produced, expressed and screened; those new promoter or began transcripts differ- with the best performance are then selected ently. In each case, PACE produced vari- and amplified. But directed evolution ants with high levels of the chosen activity is labor-intensive. Each round typically in 1.5 to 8 days. requires days to extract and clone genes Setting up PACE should be simple in and to transform cells. most biochemistry laboratories, says Liu. Generally, the more rounds of evolu- It requires standard equipment such as a tion, the better the engineered proteins, peristaltic pump, flasks and tubing. “Very so researchers must make difficult choices early on,” Liu says, “we made a philosophi- between their time and the quality of their cal decision that we would not attempt to product. Now, researchers led by David Liu create a continuous system with microflu- at Harvard University make the trade-off idics and robots and fancy machines, pre- a little less difficult. Their system, called cisely because we wanted as many scientists phage-assisted continuous evolution as possible to be able to use PACE.” (PACE), allows dozens of rounds of evo- PACE will have broad applications, says lution to occur in a single day. In essence, Liu. It can create proteins with tailor-made Nature America, Inc. All rights reserved. All rights Inc. America, Nature 1 Escherichia coli and an associated bacte- properties that might be difficult to produce rial virus, or phage, do the work of select- without hundreds or thousands of rounds

© 201 ing which genes to pass on for subsequent of evolution. Another use is to address the rounds of evolution, without the need for basic science of molecular evolution. Even any manual intervention. in this initial publication, the team showed Liu and graduate students Kevin Esvelt that the same evolutionary outcome can be and Jacob Carlson engineered a system in reached by disparate routes. In one case, which the only phage capable of replicat- two lagoons with identical starting condi- ing are those that also carry genes that pro- tions and selection pressure converged on duce desired activity. First, a crucial gene the same set of mutations, but one lagoon for phage replication is removed from the produced the mutations within 24 hours phage’s genome and placed in an ‘accessory whereas the other took 108 hours. It is easy plasmid’ in E. coli. Expression of this gene to imagine experiments that examine the can then be tied to a wide range of biologi- role of genetic drift and population size on cal activities, says Liu: recombinase activ- evolutionary outcomes, says Liu. “We can ity, polymerase activity, protein-protein replay very long evolutionary trajectories.” interactions and even protein cleavage. Thus, PACE can be used to study problems Engineered bacteria and phage are then that have so far been relegated to thought placed in vessels called lagoons, such that experiments. And doing so will not take mil- a continuous stream of E. coli move in and lions of years or even multiple ‘minipreps’. out of the vessel. The E. coli only stay in Monya Baker the lagoon for about 20 or 30 minutes, just RESEARCH PAPERS long enough for phage infection and rep- Esvelt, K.M. et al. A system for the continuous lication to occur. Only phage that activate directed evolution of biomolecules. Nature 472, the essential gene in the E. coli plasmid can 499–503 (2011).

nature methods | VOL.8 NO.6 | JUNE 2011 | 451