Team:Valencia/Project/Lab work/2 experiments

From 2008.igem.org

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<h3> Troubleshooting </h3>
<h3> Troubleshooting </h3>
In order to establish the final conditions and protocol, we have gone trough a lot of different experiments.  
In order to establish the final conditions and protocol, we have gone trough a lot of different experiments.  
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<ol>'''Lower shaking speed, larger volume and lower initial O.D.''' <br>
+
<ol>'''Low shaking speed, large volume and low initial O.D.''' <br>
At the beginning this were the conditions for our experiment. However,we did not want to add the inductor at such lower O.D., so we used to wait three hours of experiment before induction. <br>
At the beginning this were the conditions for our experiment. However,we did not want to add the inductor at such lower O.D., so we used to wait three hours of experiment before induction. <br>
*We did not obtain any encouraging results.  
*We did not obtain any encouraging results.  
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<ol>'''Increase shaking speed and reduce volume'''<br>
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<ol>'''Increased shaking speed and reduced volume'''<br>
In other to allow proper respiration the yeast culture in the LCCs, we tried and increase the revolutions per minute of of shaker. Besides, we reduced the volume for the yeast culture to have more oxygen available.
In other to allow proper respiration the yeast culture in the LCCs, we tried and increase the revolutions per minute of of shaker. Besides, we reduced the volume for the yeast culture to have more oxygen available.
*In this conditions, we obtained temperature increase for the first time.
*In this conditions, we obtained temperature increase for the first time.
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<ol>'''Higher O.D.'''
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<ol>'''Higher O.D.'''<br>
-
 
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Instead of starting the experiment at a lower O.D. and waiting several hours before inducing, we thought it would be better to start at a higher O.D. Besides, we would make sure that the O.D. at the time of induction was the same for the four strains.
 +
*In order to do that, we design the two inoculum protocol described above. With this protocol, we intend to reach an elevated O.D. without having a medium with too much glucose. We added a SP medium inoculum so as to avoid adding too much SD medium (glucose rich) to the LCC.
 +
*We obtained temperature increase and we were able to reproduce it
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Revision as of 14:43, 9 October 2008


Contents

2.-Demonstration that thermogenin-expressing yeast strains can heat their own broth medium.

Materials

In our experiments we worked with the the following Saccharomyces cerevisiae strains kindly handed by Eduardo Rial :

  • UCP+
  • UCP-
  • Gly175Δ
  • Gly76Δ

Method

Each of our experiments entails the following steps:

  1. We prepare a 30ml inoculum for each strains in 100ml Erlenmeyer flasks with SD medium. We leave it overnight in the 28ºC stove.
  2. We prepare a second inoculum for each strain, this time in with 100ml SP medium in 1L Erlenmeyer flasks. The initial O.D. of this second inoculum should be aproximately 0.2.

    The objective of this two-step protocol is to be able to start the experiment with a higher O.D. in a medium with little or no glucose. First, we need to grow our strains in a liquid medium with glucose (SD medium), in which they have a better growth rate. Second, we use SP medium both because it has a really small percentage of glucose and it is final experiment medium.

  3. We start the experiment in the LCCs with a volume of 100ml, SP medium, and a initial O.D. of 0.6.
  4. We add 1% galactose to each of the LCCs containing the four yeast strains. This sugar is the UCP gene Gal7 promoter inductor.
  5. We monitor the temperature evolution in the LCCs thanks to our LCC system.

Experiment results

Graficas, graficas, graficas...Cuando Ximo me las mande.


Troubleshooting

In order to establish the final conditions and protocol, we have gone trough a lot of different experiments.

    Low shaking speed, large volume and low initial O.D.
    At the beginning this were the conditions for our experiment. However,we did not want to add the inductor at such lower O.D., so we used to wait three hours of experiment before induction.
    • We did not obtain any encouraging results.
    • Besides, we obtained weird oscillations in most of our experiments (as described in the LCC troubleshooting).


    Medium variations
    As we were obtaining any results we desperately tried other mediums.
    • YPKAc Medium : In this medium containing acetate, yeast is suppose to only breath rather than fermenting. We try it in case the problem was that electron transport chain was inhibited.
    • Palmitate: although our Gly175Δ and Gly76Δ mutants do not need this compound, we tried and added it in case it made any difference for UCP+ strain. We tried and added it to both SP medium and YPKAc medium.
    • We did not obtain any encouraging results either.


    Increased shaking speed and reduced volume
    In other to allow proper respiration the yeast culture in the LCCs, we tried and increase the revolutions per minute of of shaker. Besides, we reduced the volume for the yeast culture to have more oxygen available.
    • In this conditions, we obtained temperature increase for the first time.
    • Besides, the weird oscillations observed before disappeared.


    Higher O.D.
    Instead of starting the experiment at a lower O.D. and waiting several hours before inducing, we thought it would be better to start at a higher O.D. Besides, we would make sure that the O.D. at the time of induction was the same for the four strains.
    • In order to do that, we design the two inoculum protocol described above. With this protocol, we intend to reach an elevated O.D. without having a medium with too much glucose. We added a SP medium inoculum so as to avoid adding too much SD medium (glucose rich) to the LCC.
    • We obtained temperature increase and we were able to reproduce it