Difference between revisions of "Team:UCLouvain/OurProject/Approach1/Results"

To assay the photocleavage of the o-nitrobenzyl tyrosine (ONB-Tyr), the previous experiment was adapted.

Two petri dishes containing M9 growth medium with ONB-Tyr were prepared. One of them was exposed to sunlight for 8 hours while the other one was kept in the dark. The cells were deposited on the petri dishes after its exposition.

The result is showed on Figure 2.

The cells used in this method are TyrA- E. coli transformed with the biobrick BBa_K577882. After transformation, the slightly reddish colonies are selected and their fluorescence is measured.

Tyr A- E. coli (not transformed), and BL21 E. coli are studied as negative controls. For each cell line, 10 mL of preculture were incubated for 12h at 37°C, in liquid LB medium. Cells are then centrifuged down (10min, 3000g). The pellets are washed 3 times with 5 mL of liquid M9 (protocol available on our website) to remove any tyrosine previously found in the preculture medium. Cells are then concentrated to an OD (optical density) value of 6 with the appropriate volume of M9 medium.

In a 12-wells plate, final volume of 3mL in each well:

• D-Tyr is added to the concentration of 0.05 mg/mL
• ONB-Tyr is added to the concentration of 0.084 mg/mL
• L-arabinose is added to the concentration of 0.1% (w/v)
• Liquid M9 is added to the volume of 3 mL.
• Add antibiotics as required

Four plates are filled as follows: tyr or ONB-Tyr, in the presence or not of arabinose are filled for the 3 types of cell (BL21, TyrA-, TyrA- + BBa_K577882). The four plates were placed under a 400WLEO / S 400-94-CR light during 0, 12, 60 and 300 minutes. Besides those plates, an ONB-Tyr toxicity control has been run, with 50/50% of Tyr/ONB-Tyr concentration with TyrA- + BBa_K577882.

After exposure, 50 µL of concentrated 6 OD cell culture is added to the well. Next, plates are placed under agitation at 37°C. Fluorescence (λexcitation = 533 nm, λemission = 588 nm) and OD are measured every 2h between 15h and 21h of incubation. The fluorescence measurements are reported to the OD measure, to normalize the results.

The graph below (Figure 4) shows the evolution of RFP fluorescence with the duration of incubation for the different samples (0, 12, 60 and 300 minutes of exposure to polychromatic light).

Figure 5 shows that the longer the exposure to polychromatic light, the higher the RFP fluorescence. When no tyrosine is available in the media (0 min of UV exposure), little fluorescence is observed. The fluorescence in the sample exposed for 300 min to light seems to have reached a stable fluorescence value after 15h of incubation.