Part:BBa_K763005

pADH2 + Gene encoding YAP1 protein

This construction has three parts:

1. A transcription factor binding site inside the promoter (UAS1),

2. A transcription factor binding site (UAS2/CSRE) and

3. The coding sequence which contains the yeast AP-1 protein (YAP1).

We used the ADH2 promoter, which is regulated by two trans-acting elements, both necessary for maximal promoter activity in the absence of a fermentable carbon source1.

When is the protein synthesized? Our construction is repressed several hundred-fold in the presence of glucose, so the transcription of YAP1 protein is initiated once the glucose in the medium is depleted.

The molecular mechanism underlying this phenomenon is as follows: there are two transcription factors which regulate the promoter, ADR1 a carbon source-sensor zinc-finger2 and CAT83. In the absence of glucose both cis-acting sites in the promoter are bound cooperatively by the transcriptional activators. ADR1 binds to the UAS1 site while CAT8 binds to the UAS2/CSRE site and regulates positively the transcription of YAP1 protein by the activation of the ADH2 promoter4. The presence of glucose downregulates the levels of the transcription factors (ADR1 and CAT8) which results in the depletion of the production of YAP1 protein by the repression of the ADH2 promoter.

Fluorescence results

This construction together with pTRR + Gene encoding ZsGreen1 construction allows yeast to express ZsGreen1 and show fluorescence when there is no glucose in the medium. To induce fluorencence, yeast must be in oxidative stress, by the addition of hydrogen peroxide.
In order to check if both constructions are functional, we induced expression using several media as depicted in this protocol. The supplemented SD media (which contains methionine and histidine but not leucine nor uracil) allows the selective growth of transgenic yeasts to obtain a big inoculant which will grow better in a glucose-rich media (YPD8%), leaving us with a culture in exponential phase and high biomass. The expression of ZsGreen1 takes place when our yeasts are deprived of glucose and there is presence of ethanol.
In order to quantify cell growth and normalize fluorescence, the DO at 600 nm of each sample was measured. Fluorescence intensity was measured at an excitation wavelength of 493 nm and an emission wavelength of 505 nm.
Figure 3 shows expression of ZsGreen1 in our transformed yeast –and not in non-transformed yeast– in YPRE broth, that is, in presence of ethanol when glucose is absent. Figure 4 shows intracellular expression of our protein. Moreover, there is such a great level of ZsGreen1 expression that it can be seen it with a naked eye, as shown in Picture 5.
Further experiments are required for a better characterization of our constructions, in which we are already working, but cannot be shown because of the lack of time.

Figure 3. Fluorescence intensity (FI) normalized by the optical density of the culture (OD) of non-transformed (control) and transformed yeast in absence of glucose and presence of ethanol.

Figure 4. A) Control of not transformed yeast. B) Intracelullar expression of ZsGreen1 in absence of glucose and presence of ethanol.

Figure 5. A non-transformed yeast (left) and a transformed yeast in induction conditions expressing ZsGreen1 (right)