Development of a synthetic biology approach to targeted directed evolution of proteins in vivo
File(s)
Author(s)
Adie, Thomas
Type
Thesis
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.
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.
Version
Open Access
Date Issued
2013-09
Date Awarded
2014-06
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Baldwin, Geoff
Freemont, Paul
Kitney, Richard
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)