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.

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 media 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 three times in YPD medium and three times 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 GAL1p 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, to obtain the overexpression effect, we tested the mRNA quantity. The results are as follows.

According to the result, we could find out that strain with yno1 overexpressed and yca1 knockout had an obvious increase of mRNAs. However, we didn’t get the same result in ndi1 overexpressed strain. We believed this result showed that the overexpression of ndi1 was failed while the yno1 one succeeded.

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

• 1. Left homologous arm of Δyca1（500bp）
• 2: Right homologous arm of Δyca1（500bp）
• 3: wide-type
• 4: wide-type

Based on the result, we determined that there was no mistake in the process of knocking out yca1 gene and that the yca1 gene was knocked out indeed.

If the growth of Δyca1 and yno1-Δyca1 was better than that of WT and yno1, it can be concluded that knocking out yca1 gene can improve yeast tolerance to H₂O₂. We diluted seed liquid to OD600 at 2, which was Saccharomyces Cerevisiae of overexpressing yno1 gene and lacking the yca1 gene (yno1-Δyca1) and strains of overexpressing yno1 gene (yno1) as control group. And then adjust the concentration of H₂O₂ in the seed liquid to 0 mM, 1 mM, and 2 mM respectively. The yeasts were cultured for 16h. Take samples every 2-4h. Its OD600 was measured by the ultraviolet spectrophotometer. OD600 reflected a growth of yeasts which can judge whether yca1 knockout can improve yeast tolerance to H₂O₂. The results are shown as follow:

As Figure.11~ Figure.14 shown, comparing the growth curves of yno1 and yno1-∆yca1 under H₂O₂ stress with 0mM, 1mM and 2mM, we can see that, the growth of strain knocking out yca1 is always better than the strain without knocking out yca1 after overexpressing yno1.

It can be concluded that knocking out yca1 can improve our engineering strain tolerance to H₂O₂.

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, also, knocking out gene yca1 can improve the tolerance to high level of ROS content.