Difference between revisions of "Team:UCLouvain/OurProject/Approach2/Results"

In order to make a UV sensitive ComR/S system three plasmids were given to us.

The promoters chosen are two proteins’ promoters induced by ComR in Streptococcus thermophilius. P1655 is a strong promoter heavily induced by ComR. On the contrary, Pshp0064 is a much weaker promoter.

The plasmids were contained inside an E. coli DH5-alpha strain that were GusA+. This meant glucoronidase's expression even when not induced. To prevent this, we extracted the plasmids and inserted them in E. coli DH5-alpha GusA- strain.

This allowed us to quantify glucuronidase activity in ComS’s presence with para-nitrophenol glucuronide (protocol described in notebook). We took the optical density at 405 nm wavelength after induction. This is the absorption wavelength of para-nitrophenol. The pcMMCD 102 and pcMMCD 106 gave no signal except background noise. Only pcMMCD 105 gave a significant signal.

To obtain a good UV detector we need the expression to be closely linked in time with the ComS presence.

We assessed this with the same test with pcMMCD 105 but this time with different incubation times. As you can see on fig.1, the signal is already visible after 20 minutes and becomes steady after 80 minutes.

Tests were also made to assess the optimal ComS concentration.

The peptide must be chemically synthetized and is therefore valuable. As such, decreasing the amount used would be economically interesting.

The ComS optimal concentration in the medium seems to be at 4 µM. Multiple experiences shows that higher concentrations even reduces the signal.

Still in order to obtain a practical product, the signal has to be clearly visible and easy to interpret. A first possibility that we tested is to grow our strains containing pcMMCD 105 on a medium with X-Gluc (5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, cyclohexylammonium salt).

X-Gluc is a glucoronidase’s substrate that produces a blue precipitate when cleaved (like X-Gal). Results showed again an optimal concentration at 4 µM of ComS 5

This system could be optimized by replacing the glucuronidase by a RFP. This would allow us to obtain a direct signal and avoid the use of expensive substrates.

Lastly, to make our system fully functional and UV sensitive, we synthetized the ComS peptide with a photocaged tyrosine (ortho-nitrobenzyl tyrosine). The photocaged tyrosine was purchased and verified by NMR. The whole peptide synthesis couldn’t be achieved. We suppose that during the synthetizing, the ortho-nitrobenzyl group moved from the phenol function to the carboxylic function. This would prevent any further amino acid addition. To overcome this problem, the use of ComR/S from Streptococcus vestibularis has been considered. Indeed this ComS version ends with a double tyrosine, getting around our current problems.

ComR/S Approach Overview ComRS_Overview