Identification of mutations that increase antibiotic resistance

Description: To better understand the process of adaptation, we are focusing on its ‘raw material’, that is the phenotypic effects of beneficial mutations. We are studying the enzyme TEM-1 β-lactamase which plays a major role in the development of antibiotic resistance. The original enzyme TEM-1 gives bacteria a high resistance to penicillin. New antibiotics (such as cefotaxime) have been developed that are able to kill bacteria with TEM-1. However, several mutations in TEM-1 increase resistance to cefotaxime. So far, we have identified 48 unique beneficial mutations that increase resistance in the first step of adaptation. In order to arrive at clinical resistance levels, second-site mutations are required and we would like (1) to identify these and (2) to compare both sets of mutations.

Used skills: Basic molecular techniques, including PCR, cloning into expression vectors, transformation to bacterial hosts, restriction and sequence analysis; MIC assays; simple bioinformatics and statistical analysis to interpret evolved enzymes.

Requirements: Molecular and Evolutionary Ecology (GEN20304) and Genetic Analyses Tools and Concepts (GEN30306) are a good preparation.

Reference: Salverda MLM et al. (2011) Initial Mutations Direct Alternative Pathways of Protein Evolution. PLoS Genet 7: e1001321.