ChBE Seminar Series: Marc Ostermeier
Tuesday, April 1, 2014
11:00 a.m.-12:00 p.m.
Room 2108, Chemical and Nuclear Engineering Building
Professor Ganesh Sriram
Professor and Vice Chair
Department of Chemical and Biomolecular Engineering
Johns Hopkins University
Mutations are central to evolution, providing the genetic variation upon which selection acts. A mutation's effect on the suitability of a gene to perform a particular function (gene fitness) can be positive, negative, or neutral. Knowledge of the distribution of fitness effects (DFE) of mutations is fundamental for understanding evolutionary dynamics, molecular-level genetic variation, complex genetic disease, the accumulation of deleterious mutations, and the molecular clock. We present a comprehensive analysis of the effect of mutation on a gene. We employed PFunkel mutagenesis to create a comprehensive codon mutagenesis library of the TEM-1 beta-lactamase gene, a gene that confers resistance to beta-lactam antibiotics such as ampicillin. We designed this library to contain 18,081 members, one for each possible codon substitution in the gene (287 positions in TEM-1 x 63 possible codon substitutions at each position). Next, we used a synthetic gene circuit that functions like a band-pass filter to partition this library into sublibraries based on an allele’s relative ability to confer ampicillin resistance (what we refer to as “gene fitness”). We quantified gene fitness by deep sequencing each sublibrary and counting the occurrence of the alleles. This DFE provides insight into the inherent benefits of the genetic code's architecture, support for the hypothesis that mRNA stability dictates codon usage at the beginning of genes, an extensive framework for understanding protein mutational tolerance, and evidence that mutational effects on protein thermodynamic stability shape the DFE. Contrary to prevailing expectations, we find that deleterious effects of mutation primarily arise from a decrease in specific protein activity and not a decrease in cellular protein levels.