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Controlling Synthetic Biological Systems at the Plasmid and Population Levels

Fedorec, Alexander Joseph Harper; (2018) Controlling Synthetic Biological Systems at the Plasmid and Population Levels. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The tools and methods for manipulating biological systems have been advancing at a great pace for the last few decades. These advances make possible the development of ever more sophisticated synthetic biological circuits for varied applications; from the bio-remediation of polluted environments to the dynamic delivery of targeted therapeutics in the body. However, the burden placed on host cells carrying these synthetic circuits leads a reduction in fitness compared to the unengineered host or wildtype environmental strain. This leads to challenges in maintaining a functioning circuit within a host population and ensuring host population existence in a competitive environment. In this thesis, I explore - and engineer - stability of synthetic biological systems at the circuit and population levels. In the first part of the thesis, I consider the population levels of multi-species bacterial communities within a chemostat environment. The difficulty with constructing such communities is that, in the absence of other interactions, differences in growth rates lead to competitive exclusion of the slower growing strain(s). I design and construct a two-species community that circumvents competitive exclusion. In this system, the slower growing strain is able to dynamically relieve that pressure by producing a bacteriocin when its population drops too low; the first system to use bacteriocins in this way. Using a mathematical model and computational simulation I show the different achievable community dynamics and demonstrate that they are easily experimentally tunable using an inducer molecule or dilution rate. The novelty of this design is that it can produce stable multi-species communities while only requiring the engineering of one of the constituents. The full system is constructed and characterised using flow cytometry and Bayesian parameter inference. Finally, the system is tested in competition in a bioreactor and shown to extend the presence of the two species. In the second part of the thesis, two approaches to enhancing the stability of plasmids in a bacterial host are demonstrated: toxin-antitoxin (TA) systems and bacteriocins. This is the first characterisation of axe/txe and microcin-V in the commensal bacteria E. coli Nissle 1917; a strain commonly used in the development of engineered probiotics. I show that both approaches improve the stability of burdensome plasmids in vitro and in an in vivo mouse tumour model. The bacteriocin is shown to have the added ability of being able to reconstitute a plasmid-bearing population if it is invaded by competitors. Novel deterministic mathematical models and Bayesian statistical methods are used to understand and explain the differences in the performance of these systems. Systems such as these will enable the deployment of synthetic biological systems in to ever more varied environments, opening doors to a great number of future applications.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Controlling Synthetic Biological Systems at the Plasmid and Population Levels
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2018. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Cell and Developmental Biology
URI: https://discovery.ucl.ac.uk/id/eprint/10063681
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