Difference between revisions of "Part:BBa K4061070"

Latest revision as of 12:38, 19 October 2021

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 fluorescence intensity of eGFP and mRFP1 in an increasing medium osmolarity. Since the reporters are regulated under promoters that respond to osmolarity changes within the cell, we would expect concentration based fluorescence readout. eGFP under OmpC promoter increased in terms of fluorescence intensity at high osmolarities while mRFP1 under OmpF promoter declined at high osmolarities and increased in low 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 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 for eGFP, while excited at 584 nm and emission read at 607 nm for mRFP1. In order to correlate the external medium concentration to intracellular osmolarity, we lysed cultured cells and measured the osmolarity of the supernatant using an osmometer. We found an exponential relationship between extracellular medium osmolarity and intracellular osmolarity. For clarity sake, we will still present data in terms of external medium concentration.

Results and Discussion

As seen in Graph 1 (n=4), the fluorescence intensity of eGFP increases 2 fold from a low concentration of 0.33M to a high concentration of 0.299M. Since eGFP is regulated by OmpC promoter, this result provides evidence that the promoter is more active at higher osmolarities.</b>

In Graph 2, we can see that OmpF regulated mRFP1 declines in intensity as the medium concentration increases. The fluorescence intensity peaks at the lowest medium concentration. Note: The fluorescence intensities for the two reporters were extremely different so we plotted them in two different graphs in order to realise the trend of fluorescence intensity with respect to medium concentration.

pOmpF and pOmpC with reporters (TCS)

Design Notes

Since this part has two regulatory units, we had to reduce chances of recombination and DNA secondary structure. So, instead of using the commonly used double terminator, we used a short sequence of T0 terminator. We used strong RBS for both fluorescence proteins to express them in high intensity important for biosensor. The eGFP is overpowering the other reporter, so teams could perhaps swap out the eGFP to any other fluorophore that they prefer.

Source

The OmpF promoter was identified from BLAST search on E. coli K12 strain. -400bp from ORF of OmpF. The other parts are taken from biobricks

References