A Continuous Culturing Device for use in Bacteriophage Evolution

Abstract

Over recent years, there has been a steady increase in bacteriophage research. This is in part due to the rise of antimicrobial resistance and in part due to our developing understanding of the role that bacteriophages play in governing microbial communities. Understanding the evolutionary dynamics of both host and phage is an important step towards the applied use of bacteriophages, be it in therapeutic settings, agriculture or aquaculture. Continuous culture techniques are a valuable tool for the systematic exploration of the variety of roles that the physical environment and the conditions within it can play in the long-term dynamics between host and phage. This thesis presents adaptions and extensions to an established turbidostat bioreactor (Gopalakrishnan et al. 2020; 2022) for use specifically in the investigation of both bacterial and viral evolution, using the well-studied model system Escherichia coli and bacteriophage T7 as proof of principle. Briefly, a bacterial host culture is maintained in the exponential phase of growth, using feedback from turbidity measurements. Host cells from this bacterial reservoir are fed on demand into an evolution chamber, which contains a mixed culture of host and phage. Both the metabolic state of the hosts in the reservoir and their influx into the evolution chamber can be controlled, allowing for a variety of evolution experiments using the bioreactor. Further to this, a second project is presented, whereby the bioreactor is adapted for use in a domestic setting, replacing specialised laboratory equipment with commonly found and inexpensive alternatives. This project, carried out in the March 2020 lockdown, describes an example experiment using dried active yeast and has potential application in an education setting. Viral life history parameters are an important method for the characterisation of bacteriophages. Lysis time and burst size, for example, can be used to evaluate the success of bacteriophage reproduction. Therefore, the final chapter of this thesis aims to demonstrate the use of one-step growth curves and Bayesian analysis to estimate the life history parameters of several bacteriophage isolates. Future research may use a combination of the aforementioned experiments to characterise the evolutionary dynamics of bacteriophages and their hosts.