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<li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Description">Description</a></li> | <li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Description">Description</a></li> | ||
<li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Design">Design</a></li> | <li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Design">Design</a></li> | ||
− | <li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/ | + | <li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Applied_Design" style="white-space:nowrap">Applied Design</a></li> |
− | <li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/ | + | <li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Experiments">Experiment</a></li> |
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− | <a class="navA" href="https://2019.igem.org/Team:Fudan-TSI/ | + | <a class="navA" href="https://2019.igem.org/Team:Fudan-TSI/Model">Model</a> |
<div class="n2"> | <div class="n2"> | ||
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<li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Model">Modeling</a></li> | <li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Model">Modeling</a></li> | ||
<li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Software">Software</a></li> | <li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Software">Software</a></li> | ||
+ | <li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Hardware">Hardware</a></li> | ||
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<li class="li2"><a class="navA2" style="white-space:nowrap;" href="https://2019.igem.org/Team:Fudan-TSI/Public_Engagement">Education & <br />Public Engagement</a></li> | <li class="li2"><a class="navA2" style="white-space:nowrap;" href="https://2019.igem.org/Team:Fudan-TSI/Public_Engagement">Education & <br />Public Engagement</a></li> | ||
− | <li class="li2"><a class="navA2" style="white-space:nowrap;" href="https://2019.igem.org/Team:Fudan-TSI/ | + | <li class="li2"><a class="navA2" style="white-space:nowrap;" href="https://2019.igem.org/Team:Fudan-TSI/Human_Practices">Integrated <br />Human Practice</a></li> |
<li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Collaborations">Collaboration</a></li> | <li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Collaborations">Collaboration</a></li> | ||
<li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Safety">Safety</a></li> | <li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Safety">Safety</a></li> | ||
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<li class="li2"><a class="navA2" style="white-space:nowrap;" href="https://2019.igem.org/Team:Fudan-TSI/Team">Team Members</a></li> | <li class="li2"><a class="navA2" style="white-space:nowrap;" href="https://2019.igem.org/Team:Fudan-TSI/Team">Team Members</a></li> | ||
− | <li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Team/ | + | <li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Attributions">Attribution</a></li> |
+ | <li class="li2"><a class="navA2" href="https://2019.igem.org/Team:Fudan-TSI/Acknowledgment">Acknowledgement</a></li> | ||
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</div> | </div> | ||
</li> | </li> | ||
− | <li class="navLi"><a class="navA noSubmenu" href="https://igem.org/ | + | <li class="navLi"><a class="navA noSubmenu" href="https://2019.igem.org/Team:Fudan-TSI/Judging">Judging</a></li> |
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− | <li class="leftNavLi"><a class="leftNavA" href="#mainTitle1">Naked eye detection</a> | + | <li class="leftNavLi"><a class="leftNavA" href="#mainTitle1">Naked eye detection</a></li> |
− | + | <li class="leftNavLi"><a class="leftNavA" href="#mainTitle2">PCR verification</a></li> | |
− | <li class="leftNavLi"><a class="leftNavA" href="#mainTitle2">PCR verification</a> | + | |
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<li class="leftNavLi"><a class="leftNavA" href="#mainTitle3">Fluorescence quantification through measurement kit</a> | <li class="leftNavLi"><a class="leftNavA" href="#mainTitle3">Fluorescence quantification through measurement kit</a> | ||
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<li class="leftNavLi"><a class="leftNavA" href="#mainTitle4">SDS-PAGE</a></li> | <li class="leftNavLi"><a class="leftNavA" href="#mainTitle4">SDS-PAGE</a></li> | ||
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<div class="col">Naked eye detection</div> | <div class="col">Naked eye detection</div> | ||
− | </div> | + | </div> |
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− | Escherichia coli (BL21) transformed with plasmid containing EGFP is | + | Single colony of Escherichia coli (BL21) transformed with plasmid containing EGFP is picked and cultured in liquid medium (2*YT). After overnight 37 ℃ culture, we transferred the liquid into an empty petri dish, and observed its fluorescence under a fluorescence microscope. Green fluorescence can be detected at the place of the bacteria solution while the rest of the plate as we expected. (Fig. 1). |
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
+ | <div class="row title1" id="mainTitle2"> | ||
+ | <div class="col">PCR verification</div> | ||
+ | </div> | ||
− | + | <div class="row para1"> | |
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− | + | We designed a set of primers which cannot amplify the nonsense mutant but is able to amplify the recovered EGFP. After PCR reaction, electrophoresis is performed and the recovered EGFP band is visibly bright while the mutant band does not appear (Fig. 2). | |
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− | + | Figure 3. Crystal structure of Cre recombinase bound to a loxP holliday junction (PDB:3MGV). | |
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</div> | </div> | ||
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− | + | <div class="col">Fluorescence quantification through measurement kit</div> | |
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</div> | </div> | ||
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− | + | After being sure that the fluorescence it recovered, we quantified its intensity with a microplate reader and the standard samples from distributed measurement kit. We followed the calibration protocol from measurement community. | |
− | + | </div> | |
− | + | </div> | |
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+ | <div class="row title2" id="mainTitle3_1"> | ||
+ | <div class="col">Cell quantity</div> | ||
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<div class="row legend"> | <div class="row legend"> | ||
<div class="row"> | <div class="row"> | ||
− | <img src="https://static.igem.org/mediawiki/2019/ | + | <img src="https://static.igem.org/mediawiki/2019/6/6c/T--Fudan-TSI--designDemo.gif" style="width:90%; margin:auto;"> |
</div> | </div> | ||
<div class="row legends"> | <div class="row legends"> | ||
− | + | Figure 6. | |
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− | For OD600 measurement, we use the silica beads in 2019 iGEM measurement kit as a standard substance. | + | For OD600 measurement, we use the silica beads in 2019 iGEM measurement kit as a standard substance.<br /><br /> |
− | As a preparation, we have measured a particle standard curve of the silica beads from maximum concentration to 0 (pure ddH2O) and used iGEM official data processing excel to generate the particle standard curve. Then, we determined the best-fitted linear region with maximum correlation coefficient R2 (Fig. | + | As a preparation, we have measured a particle standard curve of the silica beads from maximum concentration to 0 (pure ddH2O) and used iGEM official data processing excel to generate the particle standard curve. Then, we determined the best-fitted linear region with maximum correlation coefficient R2 (Fig. 3). Before each time we measure our samples, we will first measure the OD600 of the silica beads samples whose concentration are at both ends of the best-fitted linear region, which in our case, is from 300,000,000/100μl to 18,750,000/100μl for calibration of the particle standard curve. After measuring the bacteria liquid culture samples, we will change the OD600 to the number of particles according to the calibrated standard curve. |
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− | For fluorescence quantification, we use the fluorescein salt provided in 2019 iGEM measurement kit as a standard substance.< | + | For fluorescence quantification, we use the fluorescein salt provided in 2019 iGEM measurement kit as a standard substance.<br /><br /> |
− | + | As a preparation, we have measured a fluorescence standard curve of the fluorescein salt from maximum concentration to 0 (pure PBS) and used iGEM official data processing excel to generate the fluorescence standard curve. Then, we determined the best-fitted linear region with maximum correlation coefficient R2 (Fig. 4). Before each time we measure our samples, we will first measure the fluorescence intensity of the fluorescein salt samples whose concentration are at both ends of the best-fitted linear region, which in our case, is from 10μM to 0.0390625μM for calibration of the fluorescence standard curve. After measuring the bacteria liquid culture samples, we have changed the fluorescence intensity to the concentration of fluorescein salt according to the calibrated standard curve. | |
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− | Finally, we would divide the fluorescein salt concentration by the number of silica beads for our final quantified fluorescence intensity which is | + | Finally, we would divide the fluorescein salt concentration by the number of silica beads for our final quantified fluorescence intensity which is <b>c[fluorescein salt]/n[silica beads]</b> and has a unit of <b>μM/(pcs per 100 μl)</b>. |
</div> | </div> | ||
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<div class="col">SDS-PAGE</div> | <div class="col">SDS-PAGE</div> | ||
− | </div> | + | </div> |
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− | + | The EGFP nonsense mutant can only express a truncated peptide of 17.8 kD, while the full-length EGFP protein is 26.9 kD, the difference between their molecular weight could be visualized through SDS-PAGE. (Fig. 5) | |
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<span><a href="/Team:Fudan-TSI/Description" style="text-decoration:none;">Project</a></span> | <span><a href="/Team:Fudan-TSI/Description" style="text-decoration:none;">Project</a></span> | ||
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<span><a href="/Team:Fudan-TSI/Results" style="text-decoration:none;">Results</a></span> | <span><a href="/Team:Fudan-TSI/Results" style="text-decoration:none;">Results</a></span> | ||
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Revision as of 01:12, 22 October 2019