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 [http://www.cbm.uam.es/mitolab/fichapersonal.aspx?idpersona=6 Eduardo Rial] :

• UCP+ : UCP1 gene with Gal7 promoter.
• UCP- : UCP1 antisense gene with Gal7 promoter. It is used as control strain.
• Gly175Δ : UCP1 mutant sequence, Glycine 175 has been deleted.
• Gly76Δ  : UCP1 mutant sequence, Glycine 76 has been deleted. This two strains have increased uncoupling activity and do not need fatty acids to be functional.

We used plasmid pYeDP as our vector in Saccharomyces cerevisiae.

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. We leave it several hours in the 28ºC stove.
   

   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. We heat the medium and start at a temperature of aproximately 28ºC inside the LCC.
   
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.
   

Strains growth

Apart from monitoring temperature evolution, we also characterized O.D. variations of each of our strains. We took O.D. measures every one a half hours for nine hours. We carried out this experiment both in Erlenmeyer flasks in the 30ºC shaking stove and in our LCCs. This measurements were useful in order to determine some parameters for our Black Box Model. Besides, we were able to prove that the UCP was indeed being produced even though we could not see the temperature increase. Since our mutant strains Gly175Δ and Gly76Δ do not have the same growing rate as UCP+ when they are expressing the protein, the difference between the results in the strains showed that the reason for our lack of temperature increase was that we had not found the optimum conditions yet. This results made us keep on working until we obtained successful results.

Strains growth equations:

Experiment results

August_15th		Volume: 100 ml SP medium 250 rpm and slightly tilt. Initial OD: 0,2 O.D measured each one a a half hours during the first 9 hours (We observe each of the moments we opened LCCs for measurements. Induction at the beginning with galactose 1%. Plus extra shot at 9 hours. Left during two days.
September_24th		Volume: 100 ml SP medium 275 rpm and slightly tilt. Initial OD: 0,6 Induction at the beginning with galactose 1%.

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.
• We obtained temperature increase temperature increase for the first time.
• We were not able to reproduce it.
• 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.
• We tried and begin the experiment with an even higher O.D. but we have not obtained the encouraging results in yet.

Initial temperature
Even after obtaining results that shown mutants increased culture temperature, we obtained obtained weird results were none of the strains behaved as wished. We realized those experiments where the initial temperature of the culture was higher correspond to those were we obtained weird results.
• We put a lot more effort in starting at the same temperature every time.
• Nevertheless, this temperature inside the calorimeter depends on the room temperature. And room temperature can significantly change depending on the outside temperature.

Here you can see a summary of our experiments and the different conditions for them.