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

Transfo

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

Plasmid	Promoter	Reporter	Resistance
pcMMCD 102	/	Glucoronidase	Erythromycin
pcMMCD 105	P1655
pcMMCD 106	Pshp0064

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 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GUSA- strain.

This allowed us to quantify glucuronidase activity in ComS’s presence with para-nitrophenol glucuronide (protocol described in notebook). We took the optic density at 405 nm wavelength after induction. 405nm 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.

ComS (µM)	pcMMCD 102	pcMMCD 105	pcMMCD 106
0	0.078	0.495	0.077
8	0.096	1.514	0.082

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.

Test were also made to assess the optimal ComS’s concentration.

The peptide must be chemically synthetized and is therefore valuable. Decreasing the amount used would therefore be economically interesting.

The ComS’s optimal concentration in the medium seems to be at 4 µM. Multiple experiences shows this and higher concentration 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 (tyrosine ortho-nitrobenzyl). 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 acids’ 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.