Team:Valencia/Project/Lab work/2 experiments

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.

At the beginning, we set a much lower shaking speed, larger volume and lower initial O.D.
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).