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Title: A novel biological circuit to control directed evolution in vivo
Author: Mackrow, Ben Paul
ISNI:       0000 0004 9350 0812
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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Work outlined in this report describes the development a biological device which can precisely control the type, speed, and spread of mutations directed towards a target gene of interest. The intention was to continuously search through a vast DNA sequence landscape by constructing a self- controlling system in vivo, which can direct the evolution of the target protein using a non-rational approach, in an effort to identify new or improved functionality, whilst circumnavigating the need for complex and unreliable rational protein design. I describe the creation of a novel fusion protein, consisting of the mutagenic protagonist, Activation Induced Deaminase (AID), tethered to a highly targetable transcription factor (T7 RNA Polymerase). By specifically mutating only the intended target gene this system is able to increase the number of interesting mutations i.e. those which have a direct consequence on protein function. Furthermore, results show that reducing background mutations helps maintain a viable host cell. Initial results suggest that when this mutator protein is expressed in an E.coli strain with a limited base excision repair (BER) pathway, there is a ~1500 fold increase in the frequency of mutation events. Although early iterations of the system primarily showed transition mutations (C-T 75.5%, G-A 24.5%) were by far the most common, later improvements suggest a broader spectrum of mutations can be achieved by the adaptation of error-prone repair mechanisms. I also present a novel DNA assembly method named BASIC, which was designed with automation in mind, in order to streamline construction and reiteration of expression plasmids, reporter devices and the numerous fusion proteins required throughout the project. BASIC has shown exceptional performance, achieving ~90% accuracy for assemblies containing 7 DNA fragments of varying lengths. With principle mutator and construction methods in place, further work highlighted here describes the creation and characterisation of non-natural biological parts in the form of hybrid promoters/operators responsive to the inducer Tetracycline. I provide a set of variants which achieve a range of expression strengths with tight transition between uninduced (off) and induced (on) states. Estimations of relative promoter strength and critical inducer concentrations were incorporated into a deterministic model of the overall mutator circuit using a number of ordinary differential equations, including both my own parameterised kinetic functions and those taken from the literature. Although simplified, such computational models quickly revealed limitations of early circuit designs. This included the overly engineered solution to premature stoppage of the mutator device in the form of an AND gate. In its current form, the mutator system will be used to select for novel forms of the LasR molecule which can bind or associate with a number of small AHL molecules required for quorum sensing activity and activation of the downstream transcriptional activation. More specifically this could be used to 3 screen for species specific variants, and future inclusion in biosensor circuits. From a wider perspective, the mutator system could be introduced alongside alternative display screening methods depending on the target protein and its functional activity to accelerate evolution through multiple rounds of mutation and selection. Furthermore, BASIC assembly has already been widely cited by the synthetic biology community and has been integrated into the work flow of industry collaborators. It is expected that the move towards complete automation of molecular biology lab work, and the increasing integration of liquid handling platforms across biotech, the approach will become increasingly popular.
Supervisor: Baldwin, Geoff Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral