Modulating the Heat Shock Response in E. coli to Regulate Membrane Protein Expression

Membrane proteins are essential for cell survival and show potential as pharmacological and therapeutic targets in the field of nanobiotechnology.^1,2 In spite of their promise in these fields, research surrounding membrane proteins lags since their over-expression often leads to cell toxicity and death.^3,4_­ It was hypothesized that membrane protein expression could be regulated and optimized by modifying the heat shock response of Escherichia coli (E. coli). To test this hypothesis, the membrane-protein expression pathway was reprogrammed using gene-blocks that were antisense to vital membrane protein DNA and RNA binding-site sequences. aP_BAD and aHtdR gene-blocks were designed to be antisense to the DNA of the arabinose P_BAD promotor and H. turkmenica deltarhodopsin (HtdR) transmembrane protein respectively. These sequences were then employed to be cloned into a pMM102 vector and grown in NEB-5α E. coli cells. Stable glycerol stocks of the aP_BAD-PMM102 in BW25113 cells were generated for further use and testing and an aHtdR-PMM102 cell stock is still in development. After inducing the aP_BAD-PMM102 with L-arabinose to allow for membrane protein expression, spectrophotometry was used to test the optical density and growth of the cells at 600nm. The controls, with no HtdR protein coded, grew about 40% more than the aP_BAD-PMM102 cells and other test cells, as expected, and no significant difference was seen in growth between the aP_BAD-PMM102 and the other, unaltered, membrane protein-expressing cells. Continuing, the cells with membrane protein expression present as a purple pellet when spun down and as expected, only the PMM102 cells presented purple coloring with no noticeable difference in shade while the non-membrane protein-expressing cells were a yellow pellet. Further work will include cloning a GFP gene-block instead of aP_BAD or aHtdR to determine if the insignificance found in the results of this experiment is due to a flaw in the experimental design or not.

References

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