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Team:Valencia/WetLab/YeastTeam/Protocols
From 2009.igem.org
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.
