| Laboratory evolution has generated many proteins and nucleic acids with desired properties, but a single round of directed evolution typically requires days or longer with frequent intervention by the researcher. Here we describe a new platform that enables the continuous directed evolution, in principle, of any gene-encoded molecule that can be linked to the production of a protein in E. coli. During phage-assisted continuous evolution (PACE), evolving genes are transferred from host cell to host cell through a modified bacteriophage life cycle. The activity of interest is linked to phage replication through production of pIII, a protein required for phage infection. Because phage begin releasing progeny ∼10 minutes after infection and mutagenesis occurs continuously, dozens of cycles of phage replication, mutation, and selection can occur in a single day without human intervention. Using PACE, we continuously evolved T7 RNA polymerase to recognize a distinct DNA promoter and to initiate transcripts with nucleotides other than GG. Each of the three evolved activities emerged in less than one week of continuous evolution. PACE may provide solutions to otherwise intractable directed evolution problems and address novel questions in molecular evolution. |
