436951 The Distribution of Fitness Effects of Indels in TEM-1 Beta-Lactamase

Thursday, November 12, 2015: 9:50 AM
151A/B (Salt Palace Convention Center)
Courtney Gonzalez1, Paul Roberts2 and Marc Ostermeier1, (1)Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, (2)Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD

Insertions and deletions (INDELs) are an important source of genetic diversity in molecular evolution, both in nature and in the laboratory, yet a comprehensive picture of the fitness effects of INDELs is lacking. Taking advantage of innovations in mutagenesis techniques, selection schemes, and deep-sequencing technology, we created an extensive library of single codon INDELs in the TEM-1 beta-lactamase gene, which confers resistance to penicillin antibiotics, including ampicillin (Amp). The ability of E. coli cells expressing the TEM-1 protein to grow in the presence of Amp is a direct reflection of the protein’s cellular properties (e.g., kcat, Km, protein abundance), making TEM-1 beta-lactamase a convenient and well-studied model protein for sequence/structure/function studies and molecular evolution experiments. Much work has been done to characterize TEM-1, including a comprehensive map of the distribution of fitness effects of missense mutations. Our study helps to further characterize the fitness landscape of TEM-1, and adds to molecular evolution studies in general by offering an extensive picture of the distribution of fitness effects of single codon INDELs within a gene. We used PFunkel mutagenesis and inverse PCR to construct two libraries: one designed to contain all possible single codon insertions, and one with every possible single codon deletion. Using a band-pass selection scheme, each library was partitioned into sublibraries based on relative fitness, as determined by Amp resistance. Barcoded sublibraries were deep-sequenced to determine the fitness effects of individual INDELs. The distribution of fitness effects of these INDELs in TEM-1 and the relationship of fitness effects to structural features will be presented and discussed.

Extended Abstract: File Not Uploaded