Team:BIT-China/ExperimentsFeedback
To further ensure Saccharomyces cerevisiae cells will survive under a high level of hydrogen peroxide (H2O2) automatically, we made a series of genetic transformation, which will be shown as follow.
Overview As it was showed in the project, we needed to find a proper strong promoter to turn on the expression of dCas9, so we designed experiments as follow.
As Yap1 protein has over 70 downstream genes, we need to choose some gene that has known strength. Reports show that the downstream gene of Yap1 protein's mRNA level changes with and without H2O2. [1] Because we have no idea of the exact strength of these promoters, we chose both strong promoters and weak ones in this result and compared them with promoters that we have already known their strength. We tested downstream genes of Yap1 whose names were: ctt1, glr1, trx2, trr1, tsa1, sod2, gsh1, and gsh2, together with strong promoter gal1p and weak promoter msy1p. We obtained these promoters through PCR, and we ligated these promoters with GFP gene to express GFP protein and test their strength through fluorescence intensity. This is the first step to screen the proper promoter.
After confirming the proper promoter, we started to construct the plasmid contains dCas9 expression cassette and the sgRNA one.
After obtaining the promoter strength, we started to construct the dCas9-sgRNA plasmid. To avoid bringing the yeast too much living stress (every plasmid will bring living stress to the yeast), we decided to construct the dCas9 and sgRNA gene together in one plasmid. We constructed the promoter with dCas9 gene and the promoter of sgRNA, Snr52p, with the sgRNA in the plasmid as follows. The two different sgRNAs were designed to inhibit ndi1 and yno1.
We obtained the nine promoters through PCR successfully.
S1: length of GSH1p (500bp) S2: length of CTT1p (300bp) S3: length of TRX2p (275bp) S4: length of GSH2p (432bp) S5: length of FLR1p (313bp) S6: length of SOD2p (518bp) S7: Empty S8: length of TSA1p (300bp) S9: length of GLR1p (332bp) S10: length of TRR1p (544bp)
The results of fluorescence intensity are as follows.
As the experimental result of other parts, we added 2% galactose into medium, and added 1μl H2O2 before testing fluorescence intensity. We believe that the data after one hour of processing is the most reliable. We chose four promoters later, which were the strongest one, trr1p, the weakest one, glr1p, and two mild, sod2p and glr1p. We confirmed the promoter in next step of the experiment.
We respectively replaced the original promoter of dCas9 with promoters mentioned above and transformed them into yeast cells. We observed that the yeast cells with promoter trx2p grew fastest so we decided to choose trx2p as the final promoter. We tested the inhibitory effect through growth curve. We used the ndi1/yno1 overexpressed strain and obtained growth curve through its OD600. As shown by the growth curve, the growth of the strain with the dCas9-sgRNA plasmid was similar to that of the original strain, and superior to the strain overexpressing ndi1/yno1.This result proves that our feedback part works well.
[1] Gómezpastor R, Garre E, Péreztorrado R, et al. Trx2p-dependent regulation of Saccharomyces cerevisiae oxidative stress response by the Skn7p transcription factor under respiring conditions.[J]. Plos One, 2013, 8(12): e85404.
