Development of a targeted mutator system for continuous directed evolution in vivo
Author(s)
Planelles Coronas, Lucas
Type
Thesis
Abstract
In this work we pursue the development of a mutator system for directed evolution in vivo. First, we develop a comprehensive method for the evaluation of mutators. Next-generation sequencing analysis and fluctuation assays are combined to quantify mutation rates, spectrum, target specificity, sequence bias and evolutionary stability. This methodology is applied to the evaluation of various mutator candidates based on targeted transcription by T7 RNA polymerase in combination with mutagenic enzymes AID, DNA Pol IV and error-prone DNA Pol I (DNA Pol I3M). We reveal AID-T7 RNA Pol mutagenic activity mainly occurs during stationary phase and propose possible explanations for this phenomenon. Additionally, we discover a new semi-targeted mutator approach based on the coordinated action of a T7 RNA Pol mutant and error-prone DNA Pol I3M. T7K172G + Pol I3M induces all mutation types at rates three orders of magnitude above T7K172G alone and comparable to state-of-the art global mutators. We demonstrate the utility of T7K172G + Pol I3M by evolving beta-lactamase Tem-1 variants active on third-generation cephalosporin antibiotics ceftazidime and cefotaxime. Mutations within the tem1 target achieved observed gains in antibiotic resistance of 50,000-fold and 72,000-fold, respectively.
Date Issued
2024-01-11
Date Awarded
01/03/2025
License URL
Advisor
Baldwin, Geoff
Isalan, Mark
Sponsor
Biotechnology and Biological Sciences Research Council (Great Britain)
Publisher Department
Department of Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)