Team:Valencia Biocampus/Results2

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Transformation

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 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 1 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 2 shows intracellular expression of our protein.
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 1. 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 2. Intracelullar expression of ZsGreen1 in absence of glucose and presence of ethanol.