Part:BBa_K4061070

pOmpF and pOmpC with reporters (TCS)

The two component system (TCS) is a osmosensitive system where gene expression is modulated by sensing a change in intracellular osmolarity. Our composite part nicknamed "TCS basal" could be used an osmosensor mechanism where under high cellular osmolarity, green colour would be observed due to promoter OmpC regulated eGFP, while under low osmolarity, red colour will be observed due to promoter OmpF regulated mRFP1. This system was helpful for our biosensor where the target molecule (saxitoxin) induced significant osmotic changes within the cell. The Two Component System with reporter proteins formed a readout for this osmolarity changes. TCS is an attractive target for synthetic biologists as it is a modular, sensory signalling pathway. TCS could respond to environmental changes such as osmolarity, pH, temperature, phophor levels, etc. Teams working to identify both conditions possible such as high AND low pH, or high AND low osmolarity, could use our composite part which has red and green reporter proteins for effective response visualisation.

Contribution: HKUST 2021

Summary

We analysed the activity and response of OmpC promoter (BBa_R0082) to an increasing medium osmolarity. We utilised this promoter to regulate a reporter protein and observed the intensity of fluorescence as a measure of promoter activity. eGFP fluorescence intensity increased 2 fold with an increasing osmolarity which supports the hypothesis that OmpC promoter is activated at high osmolarity.

Experiments

We prepared LB growth media for E. coli BL 21 strain with varying amounts of NaCl- with final Na+ concentrations of 0.033M, 0.048M, 0.078M, 0.131M. 0.171M, 0.214M, 0.256M and finally 0.299M. Cells containing the composite part BBa_K4061070 which has OmpC regulated eGFP were cultured in the above media for 16 hours. We measured fluorescence intensity from the culture for all different concentrations along with negative-plasmid control, wild type BL 21 cells. The samples were excited at 488 nm and emission was read at 507 nm.

Results and Discussion

With increasing medium osmolarity, as a result of increasing medium Na+ concentration, the eGFP intensity is seen to increase 2 fold. The intensity increases from 10,000 AU to 20,000 AU. A negative plasmid control with WT BL21 cells is also measured for fluorescence intensity. Therefore, we can conclude that PompC is favourably activated at higher osmolarities.