Team:Valencia Biocampus/Results2

How long have you been fermenting?

In this part of the project we have achieved great results. However we are still working with our yeasts in order to improve their response and to perform a better characterisation of the constructions. To sum up, we have successfully transformed a delta-yap1 strain with the two constructions pADH2-Yap1 and pTRR2-GFP and pTRR.GFP (links), and what is more, we have observed a very neat and marked fluorescence in the transformed yeast.

Read below to follow us through fantastic results of our experimental work!

Transformation

We have successfully transformed yeast with two constructions, pADH2-Yap1 and pTRR2-GFP . We used a methionine, hisditine, uracil and leucine auxotrophic strain, to grow it up we needed a SD medium supplemented with this essential compounds. After the previously described transformations, our yeast became capable of growing up in methionine and histidine supplemented SD medium, due to we have used the two yeast plasmids Yeplac181 and Yep352 with leucine and uracil complementation, respectively.

In this figures we can see the leucine and uracil complementation wich confirms the presence of our two constructions.

Figure 1. Original (A,C) and transformed (B,D) strains, as indicated on the plates, grown up in a SD solid medium supplemented with methionine, hisditine, uracil and leucine or with methionine and hisditine (from left to right).

Figure 2. Transformed (A,C) and original (B,D) strains, as indicated on the tubes, grown up in a SD liquid medium supplemented with methionine, hisditine, uracil and leucine or with methionine and hisditine (from left to right).

Fluorescence results

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. Further information about molecular mechanisms is here.
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)