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| | </head> | | </head> |
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| − | <body id="ibody" class="scoll_dis"> | + | <body> |
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| | <ul id="left-nav"> | | <ul id="left-nav"> |
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| − | <a href="https://2018.igem.org/Team:BIT-China"><img id="imgA" class="imgA-new-pos" src="https://static.igem.org/mediawiki/2018/4/46/T--BIT-China--iGEM2018-A_img.png" /></a> | + | <a href="https://2018.igem.org/Team:BIT-China"><img id="imgA" class="imgA-new-pos" src="ht |
| − | <!-- end -->
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| − | <div id="LAB" class="LAB-container" style="margin-top:calc(25vh - 30px);">
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| − | <div id="JUN" class="collapseDiv">
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| − | <span class="collapseSpan"></span>
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| − | <a id="a-nav-dot-box-JUN">
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| − | <span class="collapseCircle"></span>JUNE</a>
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| − | <div id="collapse_body_JUN" class="collapse_body">
| + | |
| − | <div class="collapse_content">
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| − | <p class="LAB-content-p">
| + | |
| − | 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.
| + | |
| − | </p>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div id="JUL" class="collapseDiv">
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| − | <span class="collapseSpan"></span>
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| − | <a id="a-nav-dot-box-JUL">
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| − | <span class="collapseCircle"></span>
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| − | JULY
| + | |
| − | </a>
| + | |
| − | <div id="collapse_body_JUL" class="collapse_body">
| + | |
| − | <div class="collapse_content">
| + | |
| − | <h3 class="LAB-title-2">
| + | |
| − | Regulator
| + | |
| − | </h3>
| + | |
| − | <p class="LAB-content-p">
| + | |
| − | 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.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 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.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 7.13-7.16: We successfully got ESC4365 from TOP10 by Colony PCR and cultured TOP10 transformed
| + | |
| − | into ECS4365 and yno1.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 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.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 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.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h3 class="LAB-title-2">
| + | |
| − | Feedback
| + | |
| − | </h3>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 7.1-7.14 Construction of several plasmids with 9 different promoters and <i>egfp</i>.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 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.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 7.23-7.29 Construction of four other plasmid (promoters are glr1p, trr1p, tsa1p and msy1p).
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h3 class="LAB-title-2">
| + | |
| − | Output
| + | |
| − | </h3>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 7.5: Finished the codon optimization of roGFP2 gene sequences for yeast.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 7.5-7.25:Sent the sequence of roGFP2 to the company for synthesis.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 6.25-7.3:Obtained the gene of orp1 from the yeast genome.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 7.4-7.7:Finished the point mutation of orp1(C82S).
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 7.25-7.28:Synthesis the part: roGFP2-orp1 completely through OE-PCR.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 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.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 6.25-7.3:Obtained the gene of promoter from the yeast genome.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | </div>
| + | |
| − | | + | |
| − | <div id="AUG" class="collapseDiv">
| + | |
| − | <span class="collapseSpan"></span>
| + | |
| − | <a id="a-nav-dot-box-AUG">
| + | |
| − | <span class="collapseCircle"></span>AUGUST</a>
| + | |
| − | <div id="collapse_body_AUG" class="collapse_body">
| + | |
| − | <div class="collapse_content">
| + | |
| − | <h3 class="LAB-title-2">
| + | |
| − | Regulator
| + | |
| − | </h3>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 7.28-8.3:Designed the primers and Overlap-extension PCR to get
| + | |
| − | f1000-ura-r1000(ndi1),f1000-ura-r1000(yno1) successfully.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 8.3-8.16:Use colony PCR to verify if yeast endogenous gene ndi1/yno1 has been knocked out and
| + | |
| − | tested the codon optimization.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 8.19-8.29:knock out the gene yca1.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h3 class="LAB-title-2">
| + | |
| − | Feedback
| + | |
| − | </h3>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 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.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 8.6-8.12 Verification of promoter strength (pre-experimental). Yeast transformation of plasmid
| + | |
| − | with promoter flr1p.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 8.12-8.20 Pre-experiment of promoter strength verification for H2O2 gradient concentration.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 8.23-8.26 Pre-experiment of promoter strength verification.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 8.27-9.2 Design and build <i>pRS423-TEF1p-dcas9-yno1/ndi1—sgRNA</i>.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h3 class="LAB-title-2">
| + | |
| − | Output
| + | |
| − | </h3>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 7.3-8.10:Synthesis the promoter and roGFP2-orp1 completely through OE-PCR.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 8.12-8.15:Linked the promoter+roGFP2-orp1 to the plasmid pESC-Trp containing the terminator
| + | |
| − | cyc1 by restriction enzyme digestion.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 8.15-8.20:Finished the expression of roGFP2-orp1.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | | + | |
| − | </div>
| + | |
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| − | <div id="SEP" class="collapseDiv">
| + | |
| − | <span class="collapseSpan"></span>
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| − | <a id="a-nav-dot-box-SEP">
| + | |
| − | <span class="collapseCircle"></span>SEPTEMBER</a>
| + | |
| − | <div id="collapse_body_SEP" class="collapse_body">
| + | |
| − | <div class="collapse_content">
| + | |
| − | <h3 class="LAB-title-2">
| + | |
| − | Output
| + | |
| − | </h3>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 9.3-9.10: Use colony PCR to verify if yeast endogenous gene yca1 has been knocked out.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 9.13-9.18:Standardization:pGAL1-yno1-Tcyc1,pESC-ndi1,1000bp homologous+ura(â–³yno1).
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 9.21-9.30: Standardization:pGAL1-yno1-Tcyc1,1000bp homologous+ura(â–³yca1).
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h3 class="LAB-title-2">
| + | |
| − | Feedback
| + | |
| − | </h3>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 9.3-9.9 dcas9 promoter replacement
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 9.10-9.16 Redesign of the dcas9 plasmid with different promoters (promoters are
| + | |
| − | <i>TRX2p/GLR1p/TRR1p/SOD2p</i>)
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 9.17-9.23 Qpcr verified whether <i>pESC-Leu-Yno1/Ndi1</i> strain was overexpressed
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 9.24-9.30 Transfer <i>pRS423-dcas9-sgRNA-ndi1/yno1-GLR1p/TRX2p/SOD2p/TRR1p</i> into CENPK
| + | |
| − | CENPK-overexpressing <i>yno1/ndi1 </i>knockout <i>YCA1</i>.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h3 class="LAB-title-2">
| + | |
| − | Output
| + | |
| − | </h3>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 8.20-9.5:Finished the function verification of the roGFP2-orp1.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 8.20-8.28Finished the selection of promoter strength.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 8.20-9.10:Tested the codon optimization.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div id="OCT" class="collapseDiv">
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| − | <span class="collapseSpan"></span>
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| − | <a id="a-nav-dot-box-OCT">
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| − | <span class="collapseCircle"></span>OCTOBER</a>
| + | |
| − | <div id="collapse_body_OCT" class="collapse_body">
| + | |
| − | <div class="collapse_content">
| + | |
| − | <p class="LAB-content-p">
| + | |
| − | 9.10-10.5:Finished parts standardization of part: promoter, part: roGFP2-orp1, part:
| + | |
| − | promoter+roGFP2-orp1+cyc1
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 10.1-10.7 Transfer <i>pRS423-dcas9-GLR1p/TRX2p/SOD2p/TRR1p-ndi1-sgRNA</i> plasmid into
| + | |
| − | CENPK<i>-yno1-yca1-TEF2p/EN</i>.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <p class="LAB-content-p">
| + | |
| − | 10.1-10.6:Standardization:pESC-ndi1,pESC-yno1.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
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| − | <div id="PDF1" class="collapseDiv">
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| − | <span class="collapseSpan"></span>
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| − | <a id="a-nav-dot-box-PDF">
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| − | <span class="collapseCircle"></span>PDF -- Notebook Regulate</a>
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| − | <object data="https://static.igem.org/mediawiki/2018/0/08/T--BIT-China--iGEM2018-NotebookRegulate.pdf"
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| − | <span class="collapseCircle"></span>PDF -- Notebook Feedback</a>
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| − | <div class="collapse_content">
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| − | <object data="https://static.igem.org/mediawiki/2018/1/14/T--BIT-China--iGEM2018-NotebookFeedback.pdf"
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| − | <span class="collapseCircle"></span>PDF -- Notebook Output</a>
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| − | <object data="https://static.igem.org/mediawiki/2018/b/bb/T--BIT-China--iGEM2018-NotebookOutput.pdf"
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