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UBC Theses and Dissertations

Expression and purification of mouse TLR9 in HEK293F cells for use in a biosensor to detect viral and bacterial DNA Mahey, Amita


Although microbes are ubiquitous in our environments and often play crucial roles in human health, our current ability to detect them quickly and efficiently in different environments is lacking. For example, bacterial contamination of water supplies, blood platelets, and food can have serious health consequences and can even lead to death. Current strategies to detect bacterial contamination in these environments take at least 24 hours, leading to a lag time in response to the contamination. In order to more efficiently detect bacteria in these environments, we propose a biosensor to detect microbes. This biosensor will use mouse toll like receptor 9 (mTLR9), which dimerizes in the presence of bacterial and viral DNA, and can be used in setting such as hospitals, the food industry, or in homes. We will look for two signals of mTLR9 dimerization: a FRET signal from fluorophores attached to mTLR9 monomers, and electrochemical signals from redox species attached to the mTLR9 monomer via a single stranded DNA arm. The presence of these two signals will indicate a true binding event, indicative of mTLR9 dimerization, thus indicating the presence of bacteria or viruses in the tested sample. The aim of this thesis was to express and purify modified mTLR9 from HEK293F cells as a first step towards building a biosensor to detect bacterial and viral DNA. mTLR9 expression was optimized in this cell line, but mTLR9 could not be purified, as it localized to the insoluble fraction of the cell culture lysate. Future work should be aimed at expressing mTLR9 with an appropriate signal peptide and potentially expressing the protein in insect cell lines.

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