(α factor induced apoptosis)
Yeast: a-type yeast.
Function: The expression of the Fig2C promoter can be induced by adding a mating factor. We want to use this promoter to express the Bax(alpha) gene. We used the enhanced green fluorescent protein (EGFP) to test the effect of the Fig2C promoter. When the Fig2C promoter is induced, EGFP is expressed. In this way, we can detect the strengthen of the Fig2C promoter using EGFP as a reporter.
Vector construction: We first inserted the Fig2C promoter and EGFP coding sequence into the pesc-ura plasmid. And the constructed plasmid is shown in Figure 1.
Figure 1: A: pFig2C-EGFP B: pFig2C-Bax(alpha)
Functional verification: In order to verify whether α factor induces the expression of the Fig2C promoter, we transferred the constructed plasmids into yeast, and induced them with α factor. We observed the fluorescence in the transformed yeast cells under a fluorescence microscope (Figure 2). In addition, we also did quantitative PCR for EGFP mRNA. The results showed that EGFP expression significantly increased at 12 h (Figure 3), compared with the control group. These results showed that α factor can induce expression of the Fig2C promoter.
Figure 2: Fluorescence image of transformed yeast cells at 12h time
point after cultivation with (A) and without (B) 0.4 g of α factor dry powder.
Figure 3: Quantitative PCR for EGFP mRNA from Yeast with or without α factor.
Functional verification of Bax(alpha) gene
We transferred the constructed plasmid pFig2C-EGFP into yeast, we call it “Spy Yeast”, and induced them with α factor. And we define yeast that secretes alpha factor as “Killer yeast”. 10 ml of the spy yeast culture solution and 50ul of the killer yeast culture solution(OD600nm is about 1.4) were mixed for cocultivation.
Meanwhile, we used the cocultured spy yeast without integrated Bax gene and the killer yeast as a control group. Finally, we could determine that the spy yeast could be completely eliminated after 14 hours of the cocultivation (Figure 4).
Figure 4: OD(600nm) value of two experiment groups.