Date of Award

2017

Call Number

QR342 .R4 2017

Document Type

Embargoed Thesis

Degree Name

Master of Science (MS)

Department

Applied Medical Science

First Advisor

S. Monroe Duboise

Second Advisor

Stephen C. Pelsue

Third Advisor

Ah-Kau Ng

Keywords

bacteriophages, extremophile bacteria, immunology, nanogens, vaccines, LOS

Abstract

The recognition of potential biomedical applications for bacteriophages extends back to their original discovery in the early years of the twentieth century. The development of modern molecular methods has allowed for greater manipulation of hostphage genetic systems, and the conception of novel biotechnological designs.

This thesis is an exploration of potential applications for extremophile bacteria and bacteriophages isolated from alkaline hypersaline soda lakes of East Africa and California, by students and staff of the Duboise Virology and Electron Microscopy Laboratory, at the University of Southern Maine. This work is influenced by seminal genomic, molecular genetics, and structural studies made in the laboratory by Dr. Naun Lobo involving the bacteriophages Φ1N2-2 (from Lake Nakuru) and ΦOM3-ST (from Mono Lake). It builds upon Dr. Lobo’s research involving the display of potentially immunogenic parasite antigens on phage Φ1N2-2 capsid based virus like particles (VLPs). The initial design work is directed toward engineering the expression of antigens associated with Borrelia burgdorferi, the causative pathogen of Lyme disease, on the surface of the self-assembling portal protein of ΦOM3-ST. VLP-based vaccines have notably been designed, approved by FDA, and successfully used for prevention of hepatitis B and human papillomavirus infections. This thesis discusses initial stages of a VLP design for antigen display using the self-assembling, DNA packaging portal IV protein of the bacteriophage ΦOM3-ST fused in expression with an immunogenic epitope from the Outer Surface Protein A of Borrelia burgdorferi. The design of the construct is intended to allow for the relatively simple addition, substitution, or modification of the construct using basic molecular cloning methods. The engineering of bacteriophages for vaccines and other nanomedicine applications are promising, but will often depend upon having well developed genetic manipulation methods for both host bacteria and for specific bacteriophages. Thus further work began, under the guidance of Dr. Lobo, to develop the basic host-phage genetic manipulation tools for these haloalkaliphic systems. Preliminary efforts were made to design transformation and transfection protocols suitable for inducing the bacterial hosts of Φ1N2-2 and ΦOM3-ST to accept foreign DNA. Protocols intended to generate recombinant bacteriophages, and allow for the expression of recombinant proteins in the host bacterium. The uptake of Φ1N2-2 genomic DNA, with low efficiency, was demonstrated with production of phage plaques in the transfected cells. Further experiments are needed to develop optimal protocols for transforming haloalkaliphilic host bacteria with plasmid DNA, and for the transfection of ΦOM3-ST genomic DNA into the host bacterium, OM3.

In a separate experimental study, a promising diagnostic tool with many clinical and field applications was used as a potential environmental virology method for detection of specific viral sequences in environmental samples. Loop Mediated Isothermal Amplification (LAMP) is a highly sensitive assay capable of amplifying large quantities of a target DNA in an isothermal reaction producing a detectable product in as little as 15 minutes. The development of a successful LAMP assay could be used to specifically detect bacteriophages or other viruses of interest in environmental samples. Due to LAMP’s high sensitivity (it can detect as few as six copies of DNA) it can be prone to contamination and specificity problems making LAMP assay implementation V difficult. Thus further work is needed for success in use of LAMP in the context of this thesis project.

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