Team:Valencia/WetLab/YeastTeam/Protocols

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== '''Protocol used to make our yeasts produce light''' ==
== '''Protocol used to make our yeasts produce light''' ==
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PROTOCOLO DE MEDIDA DE INCREMENTOS DE CALCIO
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'''PROTOCOL OF CITOPLASMATIC Ca2+ INCREASEMENT MEASUREMENT IN ''S. cerevisiae'''''
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CITOPLASMÁTICO EN S. cerevisiae
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Modificado del protocolo seguido en Denis and Cyert (2002) JCB 156; 29-34.
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Material:
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- Plásmido pEVP11[AEQ] para la expresión de apoaequorina (Batiza et al.
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(1996) J.Biol.Chem. 271: 23357-62).
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- Solución de Coelenterazina: Coelenterazina diluida hasta 590μM en metanol
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saturado con nitrógeno. Este compuesto es altamente fotosensible y sensible
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oxígeno. Guardar a –20ºC.
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Nota: Compramos Coelenterazine, Native (CLZn) 50 μg Ref. C-2230 de
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SIGMA. Hay que gasear el metanol durante unos 5 minutos con N2 y añadir
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inmediatamente 200μL a los 50μg de coelenterazina.
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- Luminómetro: Berthold LB9507 conectado a un computador.
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- Tubos para el luminómetro.
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Procedimiento:
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1. Transformar las células con el plásmido pEVP11[AEQ].
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2. Picar transformantes y crecer o/n hasta fase estacionaria en el medio apropiado
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para mantener la expresión del plásmido.
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3. Medir OD a 660nm y calcular el volumen necesario para tener en 250μL una
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OD final de 1,8. Pasar a un tubo eppendorf con un agujerito en la tapa dicho
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volumen del cultivo.
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4. Centrifugar a 13000rpm durante 1 minuto a temperatura ambiente.
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5. Eliminar el sobrenadante.
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6. Resuspender el pellet en 250μL de medio nuevo que contenga 2μM de
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coelenterazina (aprox. 3,5μL de la solución de coelenterazina / μL de medio).
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7. Incubar durante 5,5 horas los cultivos a temperatura ambiente, en agitación y en
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la oscuridad.
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8. Justo antes de realizar la medida centrifugar el tubo 1 minuto a 13000rpm a
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temperatura ambiente, eliminar el sobrenadante y resuspender en medio nuevo
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sin coelenterazina (el volumen de medio dependerá del tipo de inducción que se
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desee).
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9. Esperar 15 min (la luminiscencia de las células se incrementa debido a que la
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glucosa induce un pico de calcio tal y como se describe en Nakajimashimada et
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al. (1991) PNAS 88; 6878-82).
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10. Medir la luminiscencia basal durante unos 15 segundos.
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11. Inyectar el volumen necesario de reactivo para provocar la inducción deseada.
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En el caso de inducción por pH alcalino:
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8. Añadir 173μL de medio
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9. a) Separar 3μL de cultivo y fijarlas por dilución en 27μL de medio con
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formaldehido al 7% (35μL de formaldehido 37% en 500μL medio). Guardar a
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4ºC para realizar contaje de células para normalizar los resultados entre
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diferentes cepas.
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b) 170μL restantes: esperar 15 minutos (pico de glucosa).
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10. Pasar los 170μL a tubos del luminómetro y medir la luminiscencia basal
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durante 15 segundos.
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11. Inyectar 30μL de KOH 100mM.
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Otros tipos de estrés:
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NaCl: 30μL NaCl 5M (0,75M final).
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CaCl2: 30μL CaCl2 1.33M (200mM final).
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KCl: 30μL KCl 100mM.
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Nota: Se deben tratar las células secuencialmente y exactamente de la misma manera
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para tener resultados reproducibles.
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          <td>[https://2009.igem.org/Team:Valencia/TeamVal https://static.igem.org/mediawiki/2009/2/20/PolaroidTeam.png]</td>
 
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">Modified from the original Denis and Cyert (2002) JCB 156; 29-34.
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          <td><html><a href="https://2009.igem.org/Team:Valencia/Project" target="_blank"> <img src="https://static.igem.org/mediawiki/2009/8/89/Logo_banner.jpg" width="237" height="136"></a></html></td>
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">Material:
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*<span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> pEVP11[AEQ] plasmid: apoaequorina expression (Batiza et al.(1996) J.Biol.Chem. 271: 23357-62).
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-->
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*<span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> Coelenterazine solution: Diluted coelenterazine until 590μM in satured N2 metanol. This compound is extremely photosensible and it's inhibited by O2. Keep at –20ºC.
 +
 
 +
<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">Note: We bought Coelenterazine, Native (CLZn) 50 μg Ref. C-2230 de
 +
<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">SIGMA. We put N2 gas into metanol during 5 minutes, and we added inmediately 200μL to the 50μg of coelenterazine.
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*<span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> Luminometer.
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*<span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> Luminometer tubes and ELISA plaques.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''Procedure:'''
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<div align="justify" style="position:relative; top:-5px; left:70px; width:500px">
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''1.''' We recieved pEVP11[AEQ] aequorin transformed yeast from Joaquin Arinyo.
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 +
 
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''2.''' We let growing up o/n in SD lacking Leu medium to maintain plasmid expression.  
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 +
 
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''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.
 +
 
 +
 
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''4.''' Centrifugate 1 minute at 13000rpm.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''5.''' Discard the supernatant.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''6.''' Resuspend the pellet into 250μL of fresh medium with coelenterazine 2μM (aprox. 3,5μL of coelenterazinestock solution / μL de medio).
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''7.''' Incubate during 5,5 horas at ambient temperature, in agitation and keeping in the darkness.
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 +
 
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''8.''' Centrifugate 1 minute at 13000rpm. Discard the supernatant and resuspend in SD lacking Leu fresh medium without coelenterazine (see the proper volum below *).
 +
 
 +
 
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''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).
 +
 
 +
 
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''10.''' Measure basal luminiscence during 15 minutes.
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 +
 
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''11.''' Add the correct reactive volum to induce luminiscence.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">In the chase of alcaline induction:
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''8.''' *Add 170μL of medium.
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 +
 
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''9.''' Add 30μL of KOH 100mM.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">Other stress types:
 +
 
 +
<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">NaCl: 30μL NaCl 5M (0,75M final).
 +
 
 +
<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">CaCl2: 30μL CaCl2 1.33M (200mM final).
 +
 
 +
<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">KCl: 30μL KCl 100mM.
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 +
<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">Note: yeasts should be treated secuentialy and in the same way to obtain reproducible results.
 +
 
 +
<div style="position:absolute; top:150px; left:580px; overflow:hidden;">

Latest revision as of 16:52, 18 October 2009














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.

Retrieved from "http://2009.igem.org/Team:Valencia/WetLab/YeastTeam/Protocols"