Departments of Biomedical Engineering, Chemistry, Molecular Biology and Biochemistry University of California, Irvine
Abstract: We recently developed an orthogonal DNA replication (OrthoRep) system in the yeast Saccharomyces cerevisiae. OrthoRep exploits an unusual selfish DNA element consisting of a DNA plasmid–DNA polymerase pair that stably replicates in the cytoplasm of yeast. The spatial and mechanistic isolation of OrthoRep from genomic replication may have fundamental significance for protein engineering and synthetic biology. For protein engineering, it offers a general platform for rapid continuous evolution where genes encoded on the OrthoRep system can be made to undergo accelerated mutation without increasing the genomic mutation rate. For synthetic biology, OrthoRep is a platform that should enable the bottom-up construction of synthetic genetic and replication systems in vivo. In this talk, I will describe our recent systematic efforts to engineer our orthogonal DNA polymerase responsible to reach a range of high mutation rates, as well as the utility of targeted rapid mutagenesis in general. I will also comment on the importance of building orthogonal central dogma components for protein engineering, directed evolution, and synthetic biology.