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

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Revision as of 21:15, 15 October 2018

JUNE

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.

JULY

Regulator

7.3-7.8: Test: cultured yeast transformed into ndi1 plasmid(yca1 gene knockout) and test its ROS 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,tnen 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 and culture.

7.20-7.24:Test:cultured yeast transformed into ndi1,ECS4365,yno1 plasmid(yca1 gene knockout)in YPD plate with 1% glucoseand 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.

AUGUST

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.

SEPTEMBER

Output

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(△yno1).

9.21-9.30: Standardization:pGAL1-yno1-Tcyc1,1000bp homologous+ura(△yca1).

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.

OCTOBER

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.