Team:BIT-China/Notebook
6.7-7.4: We got ndi1 gene fragment by using PCR and constructed ndi1-pESC-Amp-Leu plasmid successfully and designed and finished some validation experiment.
Regulator
7.3-7.8: Test: cultured yeast transformed into ndi1 plasmid(yca1 gene knockout) and test its ROS level after 24h and 48h by Measuring the fluorescence strength.
7.9-7.12: Obtain the gene of yno1 from the yeast genome and Overlap-extension PCR to connect yno1 with pESC,then we transformed DNA fragment into TOP10 pESC-yno1-Amp-Leu plasmid was extrated from escherichia coli.
7.13-7.16: We successfully got ESC4365 from TOP10 by Colony PCR and cultured TOP10 transformed into ECS4365 and yno1.
7.17-7.20: Overlap-extension PCR to connect yno1 with pESC,ndi1,and we got pESC-ndi1,pESC-yno1,pESC-ESC4365 successfully,then transformed them into TOP10 .
7.20-7.24:Test:cultured yeast transformed into ndi1,ECS4365,yno1 plasmid(yca1 gene knockout)in YPD plate with 1% glucose and different concentration of galactose(0%,1%,2%) and test its ROS after 24h and 48h by Measuring the fluorescence strength.
Feedback
7.1-7.14 Construction of several plasmids with 9 different promoters and egfp.
7.15-7.22 Transformation of four plasmids (promoters are GSH1p, GSH2p, TRX2p and FLR1p). The fluorescence intensity of all six transformed yeasts was determined.
7.23-7.29 Construction of four other plasmid (promoters are GLR1p, TRR1p, TSA1p and MSY1p).
Output
7.5: Finished the codon optimization of roGFP2 gene sequences for yeast.
7.5-7.25:Sent the sequence of roGFP2 to the company for synthesis.
6.25-7.3:Obtained the gene of orp1 from the yeast genome.
7.4-7.7:Finished the point mutation of orp1(C82S).
7.25-7.28:Synthesis the part: roGFP2-orp1 completely through OE-PCR.
7.25-8.6:Screened the promoter to find the appropriate strength of the promoter to turn on the expression of the fusion protein gene.
6.25-7.3:Obtained the gene of promoter from the yeast genome.
Regulator
7.28-8.3:Designed the primers and Overlap-extension PCR to get f1000-ura-r1000(ndi1),f1000-ura-r1000(yno1) successfully.
8.3-8.16:Use colony PCR to verify if yeast endogenous gene ndi1/yno1 has been knocked out and tested the codon optimization.
8.19-8.29:knock out the gene yca1.
Feedback
7.30-8.5 Construction of one plasmids (promoter is gal1p).Yeast transformation of three plasmids (promoters are gal1p, trr1p, tsa1p, glr1p and trx2p). Design primers for standardization.
8.6-8.12 Verification of promoter strength (pre-experimental). Yeast transformation of plasmid with promoter flr1p.
8.12-8.20 Pre-experiment of promoter strength verification for H2O2 gradient concentration.
8.23-8.26 Pre-experiment of promoter strength verification.
8.27-9.2 Design and build pRS423-TEF1p-dcas9-yno1/ndi1—sgRNA.
Output
7.3-8.10:Synthesis the promoter and roGFP2-orp1 completely through OE-PCR.
8.12-8.15:Linked the promoter+roGFP2-orp1 to the plasmid pESC-Trp containing the terminator cyc1 by restriction enzyme digestion.
8.15-8.20:Finished the expression of roGFP2-orp1.
Regulator
9.3-9.10: Use colony PCR to verify if yeast endogenous gene yca1 has been knocked out.
9.13-9.18:Standardization:pGAL1-yno1-Tcyc1,pESC-ndi1,1000bp homologous+ura(Δyno1).
9.21-9.30: Standardization:pGAL1-yno1-Tcyc1,1000bp homologous+ura(Δyca1).
Feedback
9.3-9.9 dcas9 promoter replacement
9.10-9.16 Redesign of the dcas9 plasmid with different promoters (promoters are TRX2p/GLR1p/TRR1p/SOD2p)
9.17-9.23 Qpcr verified whether pESC-Leu-Yno1/Ndi1 strain was overexpressed
9.24-9.30 Transfer pRS423-dcas9-sgRNA-ndi1/yno1-GLR1p/TRX2p/SOD2p/TRR1p into CENPK CENPK-overexpressing yno1/ndi1 knockout YCA1.
Output
8.20-9.5:Finished the function verification of the roGFP2-orp1.
8.20-8.28Finished the selection of promoter strength.
8.20-9.10:Tested the codon optimization.
9.10-10.5:Finished parts standardization of part: promoter, part: roGFP2-orp1, part: promoter+roGFP2-orp1+cyc1
10.1-10.7 Transfer pRS423-dcas9-GLR1p/TRX2p/SOD2p/TRR1p-ndi1-sgRNA plasmid into CENPK-yno1-yca1-TEF2p/EN.
10.1-10.6:Standardization:pESC-ndi1,pESC-yno1.