Team:Valencia Biocampus/Yeast

THE IDEA

Our aim in this part of the project is to detect when the yeast starts to ferment. At the end of the project we will be capable of “asking” the yeast if there is still any glucose in the media or not by the addition of H2O2. Furthermore we will be able to know how long the media has ran out of glucose. In conclution, this project allows us to know how much time has elipsed since the fermentation began.

To do this, we are going to use two gene constructions:

The ADH2 promoter fused to the YAP1 protein coding sequence. The protein YAP1 is a yeast transcription factor regulator of H2O2 adaptative response. It is stored in the citoplasm in normal conditions and, in presence of H2O2, is transported to the nucleous actting as a transcription factor. The ADH2 promoter is activated in abscence of glucose.

So, complete disappearence of glucose [] the production of YAP1 in the citoplasm whose concentration increases if the lack of glucose continues (we work with delta-yap1 strain).

The TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence. The green fluorescent protein can be detected by fluorencent emission. The tiorredoxin reducase promoter is activated by two transcriptional factors (YAP1 and SKN7 in the oxidative form), both only bind to the promoter if H2O2 is previously added to the media.

MOLECULAR MECHANISMS

Click on each plasmid to learn how our constructions work!

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.