Building an Orthogonal Replication System for Performing Directed Evolution in Escherichia coli: A Strategic Review and a Summary of the Initial Steps in Cloning Bacteriophage T7 gp4 Primase/Helicase

Techniques for directed evolution are commonly used in industry to induce mutations in a gene of interest (GOI). Rapid mutagenesis of a GOI within a host cell can be used to expedite the process of directed evolution. A system that replicates independently of the host genome is termed orthogonal. An orthogonal replication system can use an engineered error prone DNA polymerase recognizing a specific origin sequence to induce rapid mutagenesis of a GOI. The 2014 University of British Columbia (UBC) iGEM team has designed a strategy using the bacteriophage T7 replisome to introduce an orthogonal replication system into Escherichia coli. iGEM UBC have cloned and expressed three of the four genes required for T7 replication (gp1, gp2.5, and gp5). Cloning the fourth gene has been problematic; repeated mutations have been detected in a specific region of the constructs containing gp4 primase/helicase. In this study, we have attempted to correct a Gly313à Val mutation in one construct but were unsuccessful. Literature review suggests that gp4 primase/helicase expression in E. coli is toxic, which may explain the root cause of these mutations as they may alter the structure of the helicase domain. The toxicity may also be due to the presence of two proteins from the same coding region of gp4: 4A and 4B. A review of orthogonal replication systems, the current strategy to engineer an orthogonal replication system in E. coli, structural analysis of gp4 and our results suggest that mutations in the gp4 constructs may have been selected due to the toxic effects associated with the expression of the helicase domain in E. coli.