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Apr 10, 2011

Scientists Report Rapid Phage-Based Approach to Directed Molecular Evolution

  • Scientists claim to have developed a new phage-based approach to biomolecular evolution that is about 100 times faster than conventional laboratory evolution techniques and far less labor intensive. The new system, called phage-assisted continuous evolution, or PACE, is based on the continuous culture and selection of the M13 filamentous bacteriophage commonly used in phage display.

    The phage is instructed genetically to evolve in a directed manner and infects E. coli host cells, which produce the new proteins or nucleic acids. The Harvard University team that has developed the technique claims it will allow scientists to rapidly generate new proteins or nucleic acids with desired properties. Led by David R. Liu, Ph.D., professor of chemistry and chemical biology and an investigator at the Howard Hughes Medical Institute, the researchers report their work in Nature in a paper titled “A system for the continuous directed evolution of biomolecules.”

    PACE essentially comprises a system by which E. coli cells continuously flow through a fixed-volume vessel, or lagoon, which contains a replicating population of phage DNA vectors (the selection phage) encoding the gene or genes of interest. The system achieves continuous selection by linking the desired activity to the production of infectious progeny phage containing the evolving gene(s). Importantly, the average time that the host cells reside in the lagoon is less than the time required for E. coli replication. This means that mutations only accumulate in the evolving selection phage population, the authors note.

    Moreover, the speed of the phage lifecycle means that progeny phage production starts about 10 minutes after infection of the bacteria, so PACE can effectively mediate many generations of selective phage replication much faster than other laboratory evolution approaches. “Dozens of rounds of evolution can occur in a single day of PACE without human intervention,” the authors claim.

    The Harvard team demonstrated the utility of PACE in three separate experiments to evolve T7 RNA polymerase (RNAP) variants that either recognized the T3 promoter instead of T7 or initiated transcripts with ATP or CTP instead of GTP. They claim that in one experiment the system generated an enzyme with the desired target activity within a week and achieved up to 200 rounds of protein evolution in that timeframe.

    Dr. Liu states that conventional laboratory evolution methods would have taken years to complete that many rounds of evolution. “The three PACE experiments executed 45–200 rounds of evolution in 1.5–8 days and yielded T7 RNAP variants with activities on their target promoters or templates that exceeded or matched the activity of the wild-type enzyme transcribing the wild-type T7 promoter both in cells and in vitro,” the team states.

    Crucially, the PACE system can be assembled entirely from a modest collection of commercially available equipment and does not require the manufacture of any specialized components, the researchers stress. “The ability to perform dozens of rounds of evolution each day with minimal researcher involvement implies that PACE is particularly well suited to address problems or questions in molecular evolution that require hundreds to thousands of generations or the execution of many evolution experiments in parallel,” they note.

    “PACE represents the integration and manipulation of many protein and nucleic acid components in a living system to enable the rapid generation of biomolecules with new activities, a significant example and goal of synthetic biology.”


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