MacDonald, Paul Richard (2018) Directed evolution of bacteriophage T7 receptor tropism. PhD thesis, University of Warwick.

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Abstract

Bacteriophage (phage) typically infect a narrow range of hosts. In this thesis, we try to control phage-host specificity through the directed evolution of phage receptor binding domains. As a proof of concept, we aim to re-adapt the binding affinity of T7 phage so that it no longer requires lipopolysaccharide (LPS) for the infection of E. coli and instead utilises only an outer membrane protein (OmpF). To this end, we remove an essential gene from T7 and complement it in the host, thus linking phage propagation with the presence or absence of the in trans essential gene. By then providing E. coli with a receptor to be targeted by T7, we can positively select for those phage that bind to strains with that receptor, or negatively select against phage that bind strains without that receptor. We begin by demonstrating that we can amend the T7 genome in vivo and, moreover, can engineer a chimeric tail fibre fused from two different phage species (T7 and Yep-phi phage). We then identify an issue with this engineering process, namely, the appearance of individually deficient T7 that can propagate through co-infection, and both model the phenomena and take steps to address it. To inform the design of the directed evolution experiments, we develop a system of delay differential equations for modelling different strategies. For our first set of evolution experiments, we show that we can effect the binding affinity of T7 phage to different LPS phenotypes, and investigate the impact of different strategies on the outcome. We subsequently make use of this knowledge to coerce T7 affinity towards the OmpF receptor and find indirect evidence of evolved T7 infecting with the OmpF receptor, in the absence of a full LPS phenotype. Finally, we model the stochastic transfer of phage between phenotypes of different receptor binding affinities.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QH Natural history Q Science > QK Botany Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Bacteriophages, Tropisms, Cell receptors, CRISPR (Genetics)
Official Date:	September 2018
Dates:	Date Event September 2018 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	Molecular Organisation and Assembly in Cells
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Jaramillo, Alfonso (Jaramillo Rosales) ; Waterfield, Nicholas R.
Format of File:	pdf
Extent:	various pagings : illustrations
Language:	eng

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