Part:BBa_K763006

pTRR + Gene encoding ZsGreen1

This construction is made up of two parts:

1. A transcriptional factor binding site inside the promoter,

2. The coding sequence which contains a synthetic fluorescent (green) protein.

The expression of the thioredoxin reductase (TRR) promoter is regulated by two transcription factors, YAP1 and SKN7, which work together in response to oxidative stress1.

When is the protein synthesized? We obtain the green fluorescent protein by the addition of hydrogen peroxide (H2O2).

The molecular mechanism underlying this phenomenon is as follows: YAP1 transcription factor is regulated by subcellular localization. Under normal conditions an exporting receptor (Crm1) escorts YAP1 from the nucleus to the cytoplasm where it is stored. In the presence of hydrogen peroxide, another protein (Hyr1) catalyzes the formation of an intramolecular disulfide bond in YAP12. This conformational change allows YAP1 to accumulate in the nucleus and to bind to the TRR promoter by a bZIP DNA-binding domain. On the other side, Skn7 transcription factor is constitutively nuclear. In the presence of hydrogen peroxide, Skn7 is able to associate with the TRR promoter. Some studies suggest that the formation of the Skn7+ yap1+ DNA complex requires more than the binding of these two transcription factor to the promoter in close proximity. In conclusion, the two transcriptional factors require only one binding site within the promoter3. Once the two proteins are bound to the TRR promoter, the transcription of the green fluorescent protein starts.

Fluorescence results

This construction together with pADH2 + Gene encoding YAP1 protein 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)