Presenter: Amanda Kreppel − Biology
Faculty Mentor(s): Nora Kearns, Calin Plesa
Session: (In-Person) Poster Presentation
Directed evolution is a method for protein engineering which allows scientists to impose novel functions on proteins through the random and progressive introduction of mutations to their encoding gene. Traditional directed evolution approaches are inefficient, alternating cycles of manual in vitro mutation and in vivo expression and selection until a desirable advancement in protein function occurs. This limits the throughput and depth at which a protein’s mutational landscape can be explored. By eliminating in vitro mutagenesis and allowing an orthogonal error-prone polymerase to replicate a gene of interest over several generations in E. coli, we are able to push the boundaries of evolution and create large libraries of desirable mutants in vivo. Here we propose DiversiPhi29, which repurposes the replication machinery of bacteriophage ɸ29 to continuously replicate a linear plasmid (pL) carrying a gene of interest in vivo independently of host replication. Once orthogonal replication of pL is established, we will implement a system capable of tuning the mutation rate of the linear construct’s replication by altering the ratio of two ɸ29 DNA Polymerases, one of which contains error-prone mutations. This approach will enable high throughput molecular evolution in the best understood host organism.