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

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., k_cat, K_m, 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.