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

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             <ul>
 
             <ul>
 
                 <li><a href="https://2018.igem.org/Team:BIT-China/Model">Overview</a></li>
 
                 <li><a href="https://2018.igem.org/Team:BIT-China/Model">Overview</a></li>
−
                 <li><a href="https://2018.igem.org/Team:BIT-China/FluorescentProbesModel">Fluorescent Probes Model </a></li>  
+
                 <li><a href="https://2018.igem.org/Team:BIT-China/FluorescentProbesModel">Fluorescent Probes Model </a></li>
−
              <li><a href="https://2018.igem.org/Team:BIT-China/H2O2DecompositionModel">H<sub>2</sub>O<sub>2</sub>
+
                <li><a href="https://2018.igem.org/Team:BIT-China/H2O2DecompositionModel">H<sub>2</sub>O<sub>2</sub>
 
                         Decomposition Model</a></li>
 
                         Decomposition Model</a></li>
−
               
 
 
                 <li><a href="https://2018.igem.org/Team:BIT-China/roGFP2-Orp1MichaelisEquationModel">roGFP2-Orp1
 
                 <li><a href="https://2018.igem.org/Team:BIT-China/roGFP2-Orp1MichaelisEquationModel">roGFP2-Orp1
 
                         Michaelis equations Model</a></li>
 
                         Michaelis equations Model</a></li>
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         <li>
 
         <li>
−
             <a>Human Practices</a>
+
             <a>HUMAN PRACTICES</a>
 
             <ul>
 
             <ul>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/HPOverview">Overview</a></li>
 
                 <li><a href="https://2018.igem.org/Team:BIT-China/Human_Practices">Integrated Human Practices</a></li>
 
                 <li><a href="https://2018.igem.org/Team:BIT-China/Human_Practices">Integrated Human Practices</a></li>
 
                 <li><a href="https://2018.igem.org/Team:BIT-China/Public_Engagement">Education & Public Engagement</a></li>
 
                 <li><a href="https://2018.igem.org/Team:BIT-China/Public_Engagement">Education & Public Engagement</a></li>
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     </ul>
 
     </ul>
  
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     <a href="https://2018.igem.org/Team:BIT-China"><img id="imgA" class="imgA-new-pos" src="ht
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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>
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    <!-- end -->
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 +
    <div id="LAB" class="LAB-container" style="margin-top:calc(25vh - 30px);">
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                <span class="collapseCircle"></span>JUNE</a>
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                <div class="collapse_content">
 +
                    <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>
 +
                </div>
 +
            </div>
 +
 
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 +
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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">
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            <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>
 +
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 +
 
 +
 
 +
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 +
 
 +
        <div id="SEP" class="collapseDiv">
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            <a id="a-nav-dot-box-SEP">
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                <span class="collapseCircle"></span>SEPTEMBER</a>
 +
            <div id="collapse_body_SEP" class="collapse_body">
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                <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">
 +
            <span class="collapseSpan"></span>
 +
            <a id="a-nav-dot-box-OCT">
 +
                <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>
 +
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 +
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                <span class="collapseCircle"></span>PDF -- Notebook Regulate</a>
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                <span class="collapseCircle"></span>PDF -- Notebook Feedback</a>
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                    <object data="https://static.igem.org/mediawiki/2018/1/14/T--BIT-China--iGEM2018-NotebookFeedback.pdf"
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Revision as of 17:22, 17 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(â–³yno1).

9.21-9.30: Standardization:pGAL1-yno1-Tcyc1,1000bp homologous+ura(â–³yca1).

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.