Burkholderia pseudomallei is the aetiological agent of melioidosis and a potential bioterror threat. Infections are difficult to treat due to extensive antibiotic resistance and there is no prophylactic vaccine available. Studies have shown that the capsular polysaccharide (CPS) of B. pseudomallei is a virulence factor, immunogen and candidate antigen for a glycoconjugate vaccine. However, polysaccharides are complex to synthesise. One approach is to genetically engineer Escherichia coli to express the CPS; however, previous attempts at cloning the CPS coding locus from B. pseudomallei into E. coli were unsuccessful. This project proposes to clone only the essential genes from B. pseudomallei and to use native E. coli mechanisms to complete CPS synthesis. This would contribute to development of a new platform for the expression of any bespoke polysaccharide in E. coli. Six biosynthetic genes for the nucleotide sugar precursor were successfully expressed in E. coli. The structure of the precursor was verified by mass spectrometry. Precursor synthesis was also performed in an in vitro microfluidics system. This minimised the quantity of substrates and enzymes required, in preparation for the characterisation of glycosyltransferases required for CPS assembly. A novel assay for characterising glycosyltransferase activity was also developed, as current available options are prohibitively expensive and require significant quantities of glycosyltransferase which are difficult to purify. Finally, plasmids for the expression of additional glycosyltransferases to link the nascent B. pseudomallei CPS to truncated polysaccharides in E. coli were constructed. The aim of this project was to contribute to the development of a platform for the expression of bespoke polysaccharides in E. coli. The CPS of B. pseudomallei was chosen as the model polysaccharide as it has a simple structure and its manufacture is desirable for use in a vaccine against melioidosis.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:761677 |
| Date | January 2016 |
| Creators | Baldwin, Victoria Mae |
| Contributors | Harmer, Nicholas J. ; Prior, Joann |
| Publisher | University of Exeter |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10871/30158 |
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