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

 
Line 197: Line 197:
 
                         7.3-7.8: Test: cultured yeast transformed into <i>ndi1</i> plasmid(<i>yca1</i> gene knockout)
 
                         7.3-7.8: Test: cultured yeast transformed into <i>ndi1</i> plasmid(<i>yca1</i> gene knockout)
 
                         and test its
 
                         and test its
−
                         ROS
+
                         ROS level
 
                         after 24h and 48h by Measuring the fluorescence strength.
 
                         after 24h and 48h by Measuring the fluorescence strength.
 
                     </p>
 
                     </p>
Line 204: Line 204:
 
                         7.9-7.12: Obtain the gene of <i>yno1</i> from the yeast genome and Overlap-extension PCR to
 
                         7.9-7.12: Obtain the gene of <i>yno1</i> from the yeast genome and Overlap-extension PCR to
 
                         connect
 
                         connect
−
                         <i>yno1</i> with pESC,tnen we transformed DNA fragment into TOP10 pESC-yno1-Amp-Leu plasmid was
+
                         <i>yno1</i> with pESC,then we transformed DNA fragment into TOP10 pESC-yno1-Amp-Leu plasmid was
 
                         extrated from escherichia coli.
 
                         extrated from escherichia coli.
 
                     </p>
 
                     </p>
Line 215: Line 215:
 
                     <p class="LAB-content-p">
 
                     <p class="LAB-content-p">
 
                         7.17-7.20: Overlap-extension PCR to connect <i>yno1</i> with pESC,<i>ndi1</i>,and we got
 
                         7.17-7.20: Overlap-extension PCR to connect <i>yno1</i> with pESC,<i>ndi1</i>,and we got
−
                         pESC-ndi1,pESC-yno1,pESC-ESC4365 successfully,then transformed them into TOP10 and culture.
+
                         pESC-ndi1,pESC-yno1,pESC-ESC4365 successfully,then transformed them into TOP10 .
 
                     </p>
 
                     </p>
  
Line 221: Line 221:
 
                         7.20-7.24:Test:cultured yeast transformed into <i>ndi1</i>,ECS4365,<i>yno1</i> plasmid(<i>yca1</i>
 
                         7.20-7.24:Test:cultured yeast transformed into <i>ndi1</i>,ECS4365,<i>yno1</i> plasmid(<i>yca1</i>
 
                         gene knockout)in
 
                         gene knockout)in
−
                         YPD plate with 1% glucoseand different concentration of galactose(0%,1%,2%) and test its ROS
+
                         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.
 
                         after 24h and 48h by Measuring the fluorescence strength.
 
                     </p>
 
                     </p>
Line 234: Line 234:
  
 
                     <p class="LAB-content-p">
 
                     <p class="LAB-content-p">
−
                         7.15-7.22 Transformation of four plasmids (promoters are gsh1p, gsh2p, trx2p and flr1p).
+
                         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.
 
                         The fluorescence intensity of all six transformed yeasts was determined.
 
                     </p>
 
                     </p>
  
 
                     <p class="LAB-content-p">
 
                     <p class="LAB-content-p">
−
                         7.23-7.29 Construction of four other plasmid (promoters are glr1p, trr1p, tsa1p and msy1p).
+
                         7.23-7.29 Construction of four other plasmid (promoters are GLR1p, TRR1p, TSA1p and MSY1p).
 
                     </p>
 
                     </p>
  
Line 255: Line 255:
  
 
                     <p class="LAB-content-p">
 
                     <p class="LAB-content-p">
−
                         6.25-7.3:Obtained the gene of orp1 from the yeast genome.
+
                         6.25-7.3:Obtained the gene of <i>orp1</i> from the yeast genome.
 
                     </p>
 
                     </p>
  
 
                     <p class="LAB-content-p">
 
                     <p class="LAB-content-p">
−
                         7.4-7.7:Finished the point mutation of orp1(C82S).
+
                         7.4-7.7:Finished the point mutation of <i>orp1</i>(C82S).
 
                     </p>
 
                     </p>
  

Latest revision as of 03:06, 18 October 2018

LAB BOOK
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 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.

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

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.

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.