Team:Valencia/WetLab/YeastTeam/Protocols

Protocol used to make our yeasts produce light
PROTOCOL OF CITOPLASMATIC Ca2+ INCREASEMENT MEASUREMENT IN S. cerevisiae

Modified from the original Denis and Cyert (2002) JCB 156; 29-34.

Material:


 * pEVP11[AEQ] plasmid: apoaequorina expression (Batiza et al.(1996) J.Biol.Chem. 271: 23357-62).


 * Coelenterazine solution: Diluted coelenterazine until 590μM in satured N2 metanol. This compound is extremely photosensible and it's inhibited by O2. Keep at –20ºC.

Note: We bought Coelenterazine, Native (CLZn) 50 μg Ref. C-2230 de SIGMA. We put N2 gas into metanol during 5 minutes, and we added inmediately 200μL to the 50μg of coelenterazine.


 * Luminometer.


 * Luminometer tubes and ELISA plaques.

Procedure:

1. We recieved pEVP11[AEQ] aequorin transformed yeast from Joaquin Arinyo.

2. We let growing up o/n in SD lacking Leu medium to maintain plasmid expression.

3. After incubation, measure OD a 660nm y calculate the necessary volum to obtain in 250μL a final OD of 1,8. Put that volum into an eppendorf tube with a hole in its tap.

4. Centrifugate 1 minute at 13000rpm.

5. Discard the supernatant.

6. Resuspend the pellet into 250μL of fresh medium with coelenterazine 2μM (aprox. 3,5μL of coelenterazinestock solution / μL de medio).

7. Incubate during 5,5 horas at ambient temperature, in agitation and keeping in the darkness.

8. Centrifugate 1 minute at 13000rpm. Discard the supernatant and resuspend in SD lacking Leu fresh medium without coelenterazine (see the proper volum below *).

9. Wait 15 min (yeast luminiscence is increased due to a peak of Ca2+ is induced by the glucose (Nakajimashimada et al. (1991) PNAS 88; 6878-82).

10. Measure basal luminiscence during 15 minutes.

11. Add the correct reactive volum to induce luminiscence.

In the chase of alcaline induction:

8. *Add 170μL of medium.

9. Add 30μL of KOH 100mM.

Other stress types:

NaCl: 30μL NaCl 5M (0,75M final).

CaCl2: 30μL CaCl2 1.33M (200mM final).

KCl: 30μL KCl 100mM.

Note: yeasts should be treated secuentialy and in the same way to obtain reproducible results.