Difference between revisions of "Team:BIT-China/ExperimentsRegulator"

After literature searching, we selected two genes, yno1 and ndi1, as candidates for overexpression to increase the accumulation of endogenous ROS. According to our data, both of these two genes can effectively increase the accumulation of endogenous ROS^[1][2].

To determine whether overexpression of ndi1 or yno1 can increase the endogenous ROS accumulation in yeast, we constructed an expression plasmid, based on pESC-Leu, in which the cloned ndi1 or yno1 is driven by gal1 promoter. Thus the target gene can be induced by galactose and repressed by glucose.

After transformed the plasmid into Saccharomyces cerevisiae, we first measured the ROS production of yeast cells cultured in non-screening or screening medium by Fluorescent microplate reader. We mixed yeast cells with DCFH-DA, which can be oxidized by ROS to become a strong green fluorescent substance DCF (dichlorofluorescein) that cannot penetrate the cell membrane.

Fluorescent microplate reader analysis showed that overexpression of either ndi1 or yno1 may cause significant ROS production as revealed by green fluorescent substance DCF, and the fluorescence intensity in ndi1-overexpressing cells was a liitle bit higher than tat in yno1-overexpressing cells.

we performed twice in YPD medium and twice in SD-Leu-deficient medium. A time period of 0h-48h was selected and detected once every four hours. After excluding some experimental operational errors, we obtained the results in the figure.

It has been shown that external NADH dehydrogenases Ndi1 and Yno1 are involved in the generation of intracellular oxidative stress, however, the results measured by the fluorescence microplate reader cannot exclude the interference of dead cells on OD₆₀₀, we therefore examined the ROS accumulation through flow cytometry.

In order to reduce the pressure on our engineered strains from the addition of plasmid and to prevent plasmid loss, we decided to introduce the gene circuit which increases the level of endogenous ROS into the yeast genome. We chose to replace the ndi1/yno1 promoter with the galactose-inducible promoter gal1 we used. Because the promoter of the endogenous ndi1/yno1 gene in yeast is a bidirectional promoter, we chose to insert the gal1 promoter upstream of the ndi1/yno1 gene by OE-PCR.

After successfully replacing the promoter of the yeast endogenous ndi1/yno1 gene, we performed another test to determine the accumulation of ROS.

To block the response of yeast to ROS, we knocked out the yeast-derived yca1 gene. Because Yca1 is the only known yeast metacaspase, to demonstrate genetically that Ndi1 functions independently of Yca1, we overexpressed Ndi1 in yca1 mutant background. Consistently,Ndi1 overexpression exacerbated cell death in the absence of Yca1. ^[3]

After knocking out gene yca1 through homologous reorganization, we diluted seed liquid to OD₆₀₀ at 2, which was yeast cells lacking the yca1 gene (Δyca1) and CENPK 2-1C wild-type (WT) yeasts. And then adjust the concentration of hydrogen peroxide in the seed liquid to 0 mM, 1 mM, 1.5 mM , and 2 mM respectively. The yeasts were cultured for four hours and its OD₆₀₀ was measured by the ultraviolet spectrophotometer. OD₆₀₀ reflected a growth of yeast which can judge whether yca1 knockout can improve yeast tolerance to hydrogen peroxide. If the growth of Δyca1 was better than that of WT, it can be concluded that knocking out yca1 gene can improve yeast tolerance to hydrogen peroxide. The results are shown as follow:

We also carried out experiments under the condition of initial OD₆₀₀ = 0.6. The results showed that when the concentration of hydrogen peroxide was 2 mM, the Δyca1 almost stopped growing, while the WT could still grow well.

Conclusion: knock out yca1 gene cannot increase the yeast tolerance to hydrogen peroxide, or even reduce the tolerance.

It was observed that the growth rate and tolerance to hydrogen peroxide Δyca1 were lower than those of WT which was contrary to our theoretical results.

To summarize briefly, overexpress ndi1/yno1 can accumulate ROS successfully.

However, knocking out gene yca1 cannot improve the t

[1] Li W, Sun L, Liang Q, et al. Yeast AMID Homologue Ndi1p Displays Respiration-restricted Apoptotic Activity and Is Involved in Chronological Aging[J]. Molecular Biology of the Cell, 2006, 17(4):1802.

[2] Rinnerthaler M, Büttner S, Laun P, et al. Yno1p/Aim14p, a NADPH-oxidase ortholog, controls extramitochondrial reactive oxygen species generation, apoptosis, and actin cable formation in yeast.[J]. Proc Natl Acad Sci U S A, 2012, 109(22):8658-8663.

[3] Cui Y, Zhao S, Wu Z, et al. Mitochondrial release of the NADH dehydrogenase Ndi1 induces apoptosis in yeast[J]. Molecular Biology of the Cell, 2012, 23(22):4373.