Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Program Name/Specialization

Integrative Biology

Faculty/School

Faculty of Science

First Advisor

Joel Weadge

Advisor Role

Supervisor

Abstract

A biofilm can be defined by a community of microbes coexisting within a self-produced protective polymeric matrix. Exopolysaccharide (EPS) is a key component in biofilms and a contributor to their virulence and pathogenicity. The cellulose bacterial synthesis complex is one such EPS system that is found in many Enterobacteriaceae,including Escherichia coli and Salmonella spp., and is responsible for the production and secretion of the EPS cellulose. BcsC is the periplasmic protein responsible for the export of the exopolysaccharide cellulose and was the focus of this research. Sequence homology comparisons and structural predictions between BcsC, and the previously characterized alginate export proteins AlgK and AlgE indicate similar roles in facilitating the translocation of EPS across the bacterial cell wall. However, there are discrepancies between the systems, such as the purpose of several additional tetratricopeptide regions (TPRs) contained within BcsC compared to AlgK. To better understand the role that BcsC plays in cellulose export structural characterization of this protein was pursued. Six protein constructs that together cover overlapping portions of BcsCs TPR region were successfully expressed and purified, four of which were further analyzed with SAXS and screened for crystal formation. SAXS data was merged with a pre-existing protein data bank file of BcsCTPR 1-6 to identify similar regions and provided conceptual renderings as to the orientation and size of the protein. Promising crystal hits from BcsCTPR 12-21and BcsCTPR 1-15 were obtained, optimized and sent for X-ray diffraction, with resolution results between 12 and 2.8 Å. A complete dataset for BcsCTPR 1-15 has since been collected and structure solution is ongoing through a combination of molecular replacement and selenomethionine (SeMet) labelling techniques. Preliminary SeMet crystals are promising, but currently appear thinner than native crystals and additional optimization may be required before suitable X-ray diffraction data can be obtained.

Convocation Year

2019

Convocation Season

Spring

Available for download on Sunday, October 04, 2020

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