Team:Valencia Biocampus/Yeast2

THE IDEA

Our aim in this part of the project is to detect when the yeast starts to ferment. To do this, we are going to use two gene constructions. The first construction consists of the ADH2 promoter fused to the YAP1 protein coding sequence. In the second construction the TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence.

We use the ADH2 promoter (alcohol dehydrogenase) to sense the glucose/ethanol concentration. When this promoter is activated, YAP1 is expressed. This protein will attach to TRR promoter (thioredoxin reductase), which will become activated if the constitutive yeast protein SKN7 is in its oxidized form and also interacts with the promoter. This activation will enable transcription of GFP. H2O2 has to be added in order to oxidize the SKN7 protein.

Outline

- We ordered the DNA constructions: pADH2-YAP1 protein and pTRR-GFP protein which comes in the bacterium plasmid pUC57.
- We already had the Yeplac181 and Yep352 yeast vectors in our laboratory.
- We carried out four transformations in E. coli, one for each DNA molecules (the two constructions and the two vectors), in order to clone them. See the Transformation Protocol Using Heat Shock .
- We obtained several E. coli colonies in four dishes and took some colonies of each DNA (two constructions and two vectors) to grow them in liquid medium overnight at 37 ºC in a shake chamber.
- The next day we extracted the DNA molecules. See the Mini-prep Protocol.
- We obtained the purified constructions (both in pUC57 plasmid) and the purified yeast vector (YEplac181 and YEp352).
- We digested the four DNA molecules with restriction enzymes EcoRI and PstI. See the digestion protocol.
- We ligated the pTRR-GFP construction with the Yep352 vector and the ADH2-YAP1 construction with the Yeplac181 vector. See the ligation Protocol.
- The day after, we transformed E.Coli with the ligation in order to amplify and store the final constructions (pTRR-GFP/Yep352 and pADH2-YAP1/YEplac181)
- We took some of the colonies to grow them in liquid medium overnight at 37ºC in a shake chamber
- The next day, we extracted the DNA. See the Mini-prep Protocol.
- After the final purified constructions were obtained, we checked it by electrophoresis and sequenced them.
- We introduced one of the DNA constructions in yeast. See the Yeast transformation protocol.
- We checked the presence of the construction by PCR. See the protocol here.
- We used the obtained yeast in that moment and transformed it with the second construction. See the Yeast transformation protocol.
- After that, we used a PCR protocol to check the presence of both constructions. In that moment, we transferred some colonies of these yeast to grow them in YPD
- We measured the fluorescence at different glucose concentrations.
- We obtained a curve relating these values.