Date of Award
2010
Document Type
Doctoral Thesis
Degree Name
Doctor of Philosophy
Department
Biological Sciences
First Advisor
Dr. Jim O'Mahony
Second Advisor
Dr. Aidan Coffey
Abstract
Given their potential as specific and natural biocontrol agents, bacteriophages and their associated proteins have become the focus of renewed interest over the last decade. This study reviews the current state of mycobacterial diseases control worldwide and presents how phage therapy and the production of recombinant bacteriophage lysis proteins can be applied as novel biocontrol strategies for mycobacteria.
Chapter 11 presents an overview of the predicted LysA and LysB proteins in all the mycobacteriophages sequenced to date and proposes an in silico 3D predictive model of the lysin B of a recently isolated and characterised mycobacteriophage. Our data also confirms the high diversity of the lysins, especially for LysB proteins which were examined both at the sequence [20] and structural [predicted 3D] levels.
The in silico prediction of the mycobacteriophage lysins is followed by a technical chapter where the cloning of active mycobacteriophages lysins is described as well as the extensive optimisation of the different steps of cloning, expression and purification of the lysins. The genes predicted to encode for Lysin A and B proteins were identified, cloned in E.coli using pQE60 vector, and expressed following IPTG induction. An optimised purification procedure by affinity chromatography is presented and activity testing was carried out to demonstrate the peptidoglycan degrading activity of TM4 LysA and the lipolytic activity of D29 LysB proteins.
The study then focused on a novel single mycobacteriophage, Ardmore, isolated in Co. Waterford in November 2006 whose 52,141bp genome was fully sequenced and annotated. This resulted in the identification of 88 ORFs of which 34 were assigned predicted functions. Finally, the last section of this work presents a brief characterisation of this phage, and describes the cloning, expression and purification of its predicted lysis genes lysA and lysB.
Recommended Citation
Henry, Marine, "Identification and Molecular Characterisation of Mycobacteriophages and their Lysis Proteins" (2010). Theses [online].
Available at: https://sword.cit.ie/allthe/465
Access Level
info:eu-repo/semantics/openAccess