(78c) A Survey of Epistasic Effects in TEM-1 Beta-Lactamase

Epistasis is the interaction between two mutations such that the effect of the mutations together differs from what one would expect based on the effect of the individual mutations.  Models and recent experiments suggest that epistatic interactions between mutations play a crucial role in molecular evolution. We exploited recent advancements in mutagenesis techniques and deep-sequencing technology to extensively test the pervasiveness and types of epistasis in TEM-1 beta-lactamase, a convenient model protein for the study of protein evolution and the fitness effects of mutations. TEM-1 confers high resistance to penicillin antibiotics such as ampicillin (Amp). Thus, when E. coli cells bearing TEM-1 are challenged to grow in the presence of Amp, gene fitness can be directly connected to organismal fitness.  Using PFunkel mutagenesis, we constructed a comprehensive codon mutagenesis library of TEM-1 beta-lactamase designed to contain all possible single codon subsitutions in TEM-1.  We used a synthetic gene circuit to partition this library based on relative fitness for Amp resistance and performed deep sequencing on the resulting sublibraries to determine the fitness effects of 96% of the amino acid mutations in TEM-1. We then introduced into this library random second codon substitutions using PFunkel mutagenesis and measured the fitness of a set of several thousand double mutants in an analogous manner. The extent and types of epistasis observed will be presented, and the implications for natural and applied evolution will be discussed.