Development of a synthetic biology approach to targeted directed evolution of proteins in vivo

Abstract

This thesis describes the development of a novel platform for targeted directed evolution, designed to operate entirely in vivo. The system comprises a fusion of T7 RNA polymerase and activation-induced deaminase (AID); targeting is achieved by the placement of the sequence of interest under the control of a T7 promoter, whereby transcription by the polymerase exposes the DNA to mutation by the AID moiety of the fusion. The localisation effect serves to target mutation to the area downstream of the promoter, and increase the rate of that mutation compared to non-directed background activity. The system, and appropriate controls and targets, are constructed and tested in a plasmid-based experimental work-fow. The targets are, further, integrated into the genome of Escherichia coli to allow high-throughput analysis of the mutation rate of a single copy target. Nucleotide sequencing is used to confrm both enhanced mutagenesis of the system, and a high degree of targeting. The system is then applied to a test case, diversifcation of a transcription factor (LasR from Pseudomonas aeruginosa, encoded by the lasR gene), with an eye to producing an orthogonal signal/response pair with promoter PLas. A logic gate-based flter is designed and constructed to allow tight moderation of a feedback loop to control the mutator, allowing it to be 'shut off' once desired function is exhibited by the target protein.