Project

In vitro evolution of antibiotic resistance

Biological organisms are of limited use for the direct experimental study of evolution, because (i) they evolve slowly due to a low mutation rate, (ii) the relationship between genotype and phenotype is complex, and (iii) the experimental control of variables that affect the evolutionary outcome is difficult.

Description:

In vitro evolution of single enzymes, where mutations can be introduced at much higher rates in the PCR machine, experimental variables (e.g. mutation, recombination, strength of selection) can be better controlled, and the genetic causes of evolved changes can be traced back to the genotype directly, provides a powerful alternative. We use in vitro evolution of resistance to various antibiotics conferred by a beta-lactamase enzym (encoded by a 861-bp TEM-1 gene) to address various fundamental and applied questions, including the evolutionary causes and consequences of sex and recombination, the evolution of genetic robustness, evolutionary trade-offs between generalist and specialist phenotypes, and the predictability of adaptation (measured as the repeatability of adaptation among independently evolving lines). As fitness measure we use the minimal inhibitory concentration (MIC) of antibiotic or competitive fitness at sub-MIC concentrations of antibiotic. Results from in vitro evolution experiments can be compared to those obtained from more realistic in vivo evolution of beta-lactamase inside a bacterial host.

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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) provide a good preparation.

Contact

Merijn Salverda and Arjan de Visser

Reference:

Weinreich et al. 2006 Darwinian evolution can follow only very few mutational paths to fitter proteins. Science 312: 111-114.