A STUDY OF TAILED PHAGES AND PHAGE BIOLOGY TO EMPOWER ACCESSIBILITY OF PHAGE-BASED TECHNOLOGIES FOR APPLICATIONS IN FOOD AND WATER SAFETY

Abstract

As the natural viral predators of bacteria, bacteriophages (“phages”) exist anywhere their hosts can grow. As obligate parasites, phage replication is completely dependent on the ability to hijack a host’s machinery and energy reserves to redirect metabolism towards viral replication. Having evolved alongside their hosts for billions of years, phages have developed a wide range of interesting morphologies and unique molecular characteristics. Scientists have redirected many aspects of phage biology to produce cutting-edge biotechnologies that aim to address an array of issues across a variety of fields. Around 95 % of all known phages belong to the Caudovirales order of tailed phages. These phages identify and bind to permissive hosts via highly specific interactions that occur between adsorptive phage proteins (i.e., tail fibers, whisker proteins, tail spikes, etc.) and targeted bacterial surface receptors. Over the past 15-20 years, the emergence of cost-effective and user-friendly engineering techniques like homologous recombination, CRISPR/Cas9, in vivo yeast assembly, and in vitro Gibson assembly has allowed for phages to be made into biosensors that serve as biological indicators of bacterial contaminants in food, water, and environmental samples. Further, if material binding tags are incorporated into phage capsids, phages can be site-specifically functionalized onto different surfaces in capsid-directed orientations. These orientations protect the phage’s adsorptive machinery from being accidentally blocked during surface conjugation. If phage-based tools are to be made globally accessible in the future, certain hurdles must first be overcome. The work described in this dissertation addresses how i.) initial phage characterization assays are still labor-intensive and should be redesigned as high-throughput assays, ii.) how the materials used in phage-conjugated technologies are too expensive for feasible industrial scaling, how iii.) tagging a phage’s capsid previously required the phage’s genome be modified, and how iv.) phage technology could benefit if isolated phage genomes were better characterized. The research presented here offers strategies to overcome these limitations and provides a critical step towards making phage-based tools more widely accessible.