Team:NCTU Formosa/CI data

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<div id="banner"><a href = "https://2011.igem.org/Team:NCTU_Formosa"><img src = "https://static.igem.org/mediawiki/2011/8/8c/Banner010.jpg" height="236" width="944"/></a></div>
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             </ul>
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         <li><a class="arrow no-click">CI promoter </a>
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             </ul>
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             </ul>
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<br><br>
<br><br>
<div id="blueBox"><p> High Temperature Induced System – cI Promoter & cI repressor
<div id="blueBox"><p> High Temperature Induced System – cI Promoter & cI repressor
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<p>To verify that this circuit can work as expectation, we use GFP (Green Fluorescent Protein, <a href=http://partsregistry.org/Part: BBa_E0040”>BBa_E0040</a>) as reporter protein. Part <a href=http://partsregistry.org/Part: BBa_K098988”>BBa_K098988</a> is also designed by Team Harvard 2008. In this circuit <a href=http://partsregistry.org/Part: BBa_K098988”>BBa_K098988</a>, we can regard GFP (Green Fluorescent Protein, <a href=http://partsregistry.org/Part: BBa_E0040”>BBa_E0040</a>) as reporter protein to tell us that the gene after previous circuit <a href=http://partsregistry.org/Part: BBa_K098995”>BBa_K098995</a> can be induced or not(Figure 3).</p>
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<p>To verify that this circuit can work as expectation, we use GFP (Green Fluorescence Protein, <a href=" http://partsregistry.org/Part:BBa_E0040">BBa_E0040</a>) as reporter protein. Part <a href=" http://partsregistry.org/Part:BBa_K098988">BBa_K098988</a> is also designed by Team Harvard 2008. In this circuit <a href=" http://partsregistry.org/Part:BBa_K098988">BBa_K098988</a>, we can regard GFP (Green Fluorescence Protein, <a href=" http://partsregistry.org/Part:BBa_E0040">BBa_E0040</a>) as reporter protein to tell us that the gene after previous circuit <a href=" http://partsregistry.org/Part:BBa_K098995">BBa_K098995</a> can be induced or not(Figure 3).</p>
<br>
<br>
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<div><img src = "http://partsregistry.org/wiki/images/d/d3/CI-3.png" width="700"></div>
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<center><div><img src = "http://partsregistry.org/wiki/images/d/d3/CI-3.png" width="700"></div></center>
<br><b> Figure 3.</b>
<br><b> Figure 3.</b>
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Part <a href=http://partsregistry.org/Part: BBa_K098988”>BBa_K098988</a> Design, with Part <a href=http://partsregistry.org/Part: BBa_K098995”>BBa_K098995</a> followed by Part <a href=http://partsregistry.org/Part: BBa_E0240”>BBa_E0240</a> (include <a href=http://partsregistry.org/Part:BBa_B0032”>BBa_B0032</a>+<a href=http://partsregistry.org/Part: BBa_E0040”>BBa_E0040</a>+<a href=http://partsregistry.org/Part: BBa_B0010”>BBa_B0010</a>+<a href=http://partsregistry.org/Part: BBa_B0012”>BBa_B0012</a>). Among this circuit, GFP (Green Fluorescent Protein, <a href=http://partsregistry.org/Part: BBa_E0040”>BBa_E0040</a>) is reporter protein to tell us that the gene after the part <a href=http://partsregistry.org/Part: BBa_K098995”>BBa_K098995</a> will be induced or not. <br><br>
+
Part <a href=" http://partsregistry.org/Part:BBa_K098988">BBa_K098988</a> Design, with Part <a href=" http://partsregistry.org/Part:BBa_K098995">BBa_K098995</a> followed by Part <a href=" http://partsregistry.org/Part:BBa_E0240">BBa_E0240</a> (include <a href=" http://partsregistry.org/Part:BBa_B0032">BBa_B0032</a>+<a href=" http://partsregistry.org/Part:BBa_E0040">BBa_E0040</a>+<a href=" http://partsregistry.org/Part:BBa_B0010">BBa_B0010</a>+<a href=" http://partsregistry.org/Part:BBa_B0012">BBa_B0012</a>). Among this circuit, GFP (Green Fluorescence Protein, <a href=" http://partsregistry.org/Part:BBa_E0040">BBa_E0040</a>) is reporter protein to tell us that the gene after the part <a href=" http://partsregistry.org/Part:BBa_K098995">BBa_K098995</a> will be induced or not. <br><br>
<p>
<p>
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First day, we incubated E.coli which contains this circuit (<a href=http://partsregistry.org/Part: BBa_K098988”>BBa_K098988</a>) with GFP (Green Fluorescent Protein, <a href=http://partsregistry.org/Part: BBa_E0040”>BBa_E0040</a>) at 37℃ overnight. The next day, we transferred them to new three tubes with M9 medium. When the O.D. reached 0.08, we incubated them to 25℃, 37℃, and 42℃, respectively. We collected the samples once an hour, diluting them 200x for accurate GFP (Green Fluorescent Protein, <a href=http://partsregistry.org/Part: BBa_E0040”>BBa_E0040</a>) measurements. Here is our experiment raw data (Table 1). The mature data will be obtained by unit-conversion according to beads control data (Table 2). We can use Table2 data to draw line chart data, which is easy for us to compare the mean fluorescence at different temperature, 25℃, 37℃, and 42℃. (Figure4) The vertical axis is mean fluorescence, and the horizontal axis is time (unit: an hour).  
+
First day, we incubated E.coli which contains this circuit (<a href=" http://partsregistry.org/Part:BBa_K098988">BBa_K098988</a>) with GFP (Green Fluorescence Protein, <a href=" http://partsregistry.org/Part:BBa_E0040">BBa_E0040</a>) at 37℃ overnight. The next day, we transferred them to new three tubes with M9 medium. When the O.D.(optical density) reached 0.08, we incubated them to 25℃, 37℃, and 42℃, respectively. We collected the samples once an hour, diluting them 200x for accurate GFP (Green Fluorescence Protein, <a href=" http://partsregistry.org/Part:BBa_E0040">BBa_E0040</a>) measurements. Here is our experiment original data (Table 1). The data after unit-conversion is shown in Table 2. We can use Table2 data to draw line chart, which is easy for us to compare the mean fluorescence at different temperatures, 25℃, 37℃, and 42℃. (Figure4) The vertical axis is mean fluorescence, and the horizontal axis is time (unit: an hour).  
</p>
</p>
<p>
<p>
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<b>Original Data (Related fluorescent unit)</b><br>
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<center><div><b>Original Data (Related fluorescent unit)</b></div><br></center>
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<table style="border: 1px dotted rgb(0, 0, 0);width: 300px; align="right" cellpadding="5" cellspacing="5" frame="border" rules="all">
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<center><table style="border: 1px dotted rgb(0, 0, 0);width: 300px; align="right" cellpadding="5" cellspacing="5" frame="border" rules="all">
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<tr><td align=“right”></td><td align="right">25℃</td><td align=“right”>37℃</td><td align=“right”>42℃</td></tr>
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<tr><td align="right"></td><td align="right">25℃</td><td align="right">37℃</td><td align="right">42℃</td></tr>
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<tr><td align=“right”>0</td><td align=“right”>9.02</td><td align=“right”>8.76</td><td align=“right”>9.14</td></tr>
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<tr><td align="right">0</td><td align="right">9.02</td><td align="right">8.76</td><td align="right">9.14</td></tr>
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<tr><td align=“right”>1</td><td align=“right”>4.66</td><td align=“right”>8.36</td><td align=“right”>17.54</td></tr>
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<tr><td align="right">1</td><td align="right">4.66</td><td align="right">8.36</td><td align="right">17.54</td></tr>
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<tr><td align=“right”>2</td><td align=“right”>2.82</td><td align=“right”>6.46</td><td align=“right”>29.24</td></tr>
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<tr><td align="right">2</td><td align="right">2.82</td><td align="right">6.46</td><td align="right">29.24</td></tr>
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<tr><td align=“right”>3</td><td align=“right”>2.37</td><td align=“right”>5.00</td><td align=“right”>26.44</td></tr>
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<tr><td align="right">3</td><td align="right">2.37</td><td align="right">5.00</td><td align="right">26.44</td></tr>
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<tr><td align=“right”>4</td><td align=“right”>1.70</td><td align=“right”>4.69</td><td align=“right”>22.27</td></tr>
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<tr><td align="right">4</td><td align="right">1.70</td><td align="right">4.69</td><td align="right">22.27</td></tr>
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<tr><td align=“right”>5</td><td align=“right”>1.80</td><td align=“right”>4.40</td><td align=“right”>21.94</td></tr>
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<tr><td align="right">5</td><td align="right">1.80</td><td align="right">4.40</td><td align="right">21.94</td></tr>
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<tr><td align=“right”>6</td><td align=“right”>1.52</td><td align=“right”>4.72</td><td align=“right”>21.57</td></tr>
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<tr><td align="right">6</td><td align="right">1.52</td><td align="right">4.72</td><td align="right">21.57</td></tr>
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<tr><td align=“right”>7</td><td align=“right”>1.58</td><td align=“right”>4.78</td><td align=“right”>21.01</td></tr>
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<tr><td align="right">7</td><td align="right">1.58</td><td align="right">4.78</td><td align="right">21.01</td></tr>
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<tr><td align=“right”>9</td><td align=“right”>1.22</td><td align=“right”>3.9</td><td align=“right”>22.21</td></tr>
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<tr><td align="right">9</td><td align="right">1.22</td><td align="right">3.9</td><td align="right">22.21</td></tr>
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</table>
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</table></center>
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<br><b> Table 1. </b> Related fluorescent unit at 25℃,37℃,42℃ in 9 hours<br><br>
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<center><br><b> Table 1. </b> Related fluorescent unit at 25℃,37℃,42℃ in 9 hours<br><br></center>
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<b>Data after unit conversion</b><br>
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<center><div><b>Data after unit conversion</b></div><br></center>
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<table style="border: 1px dotted rgb(0, 0, 0);width: 500px; align="right" cellpadding="5" cellspacing="5" frame="border" rules="all">
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<center><table style="border: 1px dotted rgb(0, 0, 0);width: 450px; align="right" cellpadding="5" cellspacing="5" frame="border" rules="all">
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<tr><td align=“right”></td><td align=“right”>25℃</td><td align=“right”>37℃</td><td align=“right”>42℃</td></tr>
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<tr><td align="right"></td><td align="right">25℃</td><td align="right">37℃</td><td align="right">42℃</td></tr>
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<tr><td align=“right”>0</td><td align=“right”>2400.9500</td><td align=“right”>2326.2350</td><td align=“right”>2435.4930</td></tr>
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<tr><td align="right">0</td><td align="right">2400.9500</td><td align="right">2326.2350</td><td align="right">2435.4930</td></tr>
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<tr><td align=“right”>1</td><td align=“right”>1175.9040</td><td align=“right”>2211.6410</td><td align=“right”>4926.7290</td></tr>
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<tr><td align="right">1</td><td align="right">1175.9040</td><td align="right">2211.6410</td><td align="right">4926.7290</td></tr>
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<tr><td align=“right”>2</td><td align=“right”>683.2785</td><td align=“right”>1673.7370</td><td align=“right”>8559.5680</td></tr>
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<tr><td align="right">2</td><td align="right">683.2785</td><td align="right">1673.7370</td><td align="right">8559.5680</td></tr>
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<tr><td align=“right”>3</td><td align=“right”>566.2294</td><td align=“right”>1268.9040</td><td align=“right”>7677.1730</td></tr>
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<tr><td align="right">3</td><td align="right">566.2294</td><td align="right">1268.9040</td><td align="right">7677.1730</td></tr>
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<tr><td align=“right”>4</td><td align=“right”>395.3901</td><td align=“right”>1184.0880</td><td align=“right”>6377.2450</td></tr>
+
<tr><td align="right">4</td><td align="right">395.3901</td><td align="right">1184.0880</td><td align="right">6377.2450</td></tr>
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<tr><td align=“right”>5</td><td align=“right”>420.5876</td><td align=“right”>1105.1540</td><td align=“right”>6160.8630</td></tr>
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<tr><td align="right">5</td><td align="right">420.5876</td><td align="right">1105.1540</td><td align="right">6160.8630</td></tr>
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<tr><td align=“right”>6</td><td align=“right”>350.3407</td><td align=“right”>1192.2770</td><td align=“right”>6160.8630</td></tr>
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<tr><td align="right">6</td><td align="right">350.3407</td><td align="right">1192.2770</td><td align="right">6160.8630</td></tr>
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<tr><td align=“right”>7</td><td align=“right”>365.3116</td><td align=“right”>1208.6670</td><td align=“right”>5988.1660</td></tr>
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<tr><td align="right">7</td><td align="right">365.3116</td><td align="right">1208.6670</td><td align="right">5988.1660</td></tr>
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<tr><td align=“right”>9</td><td align=“right”>276.2400</td><td align=“right”>970.0604</td><td align=“right”>6358.6760</td></tr>
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<tr><td align="right">9</td><td align="right">276.2400</td><td align="right">970.0604</td><td align="right">6358.6760</td></tr>
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</table>
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</table></center>
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<br><b> Table 2. </b> Molecules of equivalent fluorescence at 25℃,37℃,42℃ in 9 hours <br><br>
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<center><br><b> Table 2. </b> Molecules of equivalent fluorescence at 25℃,37℃,42℃ in 9 hours <br><br></center>
<br>
<br>
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<div><img src = "http://partsregistry.org/wiki/images/6/6e/CI-4.png" width="700"></div>
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<center><div><img src = "http://partsregistry.org/wiki/images/6/6e/CI-4.png" width="700"></div></center>                               
<br><b>Figure 4.</b><br>
<br><b>Figure 4.</b><br>
Line chart of molecules of equivalent fluorescence performance at 25℃,37℃,42℃ in 9 hours. The vertical axis is mean fluorescence, and the horizontal axis is time. Form the experimental data, we find that MEFL increases steeply at 42℃ in first two hours, which means the expression of GFP is high. Although after the first two hours its performance decreases and maintains at certain volume, it still expresses a lot more than the other two samples at 25℃,37℃. <br><br>
Line chart of molecules of equivalent fluorescence performance at 25℃,37℃,42℃ in 9 hours. The vertical axis is mean fluorescence, and the horizontal axis is time. Form the experimental data, we find that MEFL increases steeply at 42℃ in first two hours, which means the expression of GFP is high. Although after the first two hours its performance decreases and maintains at certain volume, it still expresses a lot more than the other two samples at 25℃,37℃. <br><br>
</p>
</p>
<p>
<p>
-
The following diagrams, Figure5-1.~Figure5-3., are from the Flow cytometer  experiment we did. The green fluorescent intensity of E.coli incubated at 25℃ (Figure5-1) and 37℃(Figure5-2) for 9 hours is lower than incubated at 42℃(Figure5-3). Also can refer to the data from Table 1. In Figure5-2., the green fluorescent intensity of E.coli incubated at 37℃ moves right a little bit. Last, in Figure5-3., incubated at 42℃ for 9 hours, the peak of green fluorescent intensity is significantly stronger than incubated at 25℃ and 37℃ for 9 hours.</p>
+
The following diagrams, Figure5-1.~Figure5-3., are from the Flow cytometer  experiment we did. The green fluorescence intensity of E.coli incubated at 25℃ (Figure5-1) and 37℃(Figure5-2) for 9 hours is lower than incubated at 42℃(Figure5-3),which also can refer to the data from Table 1. In Figure5-2., the peak of green fluorescence intensity of E.coli incubated at 37℃ for 9 hours moves right a little bit than incubated at 25℃ for 9 hours. Last, in Figure5-3., incubated at 42℃ for 9 hours, the peak of green fluorescence intensity is significantly stronger than incubated at 25℃ and 37℃ for 9 hours.</p>
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<br>
<div><img src = "http://partsregistry.org/wiki/images/thumb/e/ed/CI-5-1.png/800px-CI-5-1.png" width="820"></div>
<div><img src = "http://partsregistry.org/wiki/images/thumb/e/ed/CI-5-1.png/800px-CI-5-1.png" width="820"></div>
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<br><b> Figure5-1. </b> Fluorescence of sample at 25℃, 0hr &9 hour.<br><br>
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<br><b> Figure5-1. </b> Fluorescence of sample at 25℃, 0hr &9 hour. X-axis is related green fluorescence intensity, and Y-axis is cells number.<br><br>
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<div><img src = "http://partsregistry.org/wiki/images/thumb/9/98/CI-5-2.png/800px-CI-5-2.png" width="820"></div>
<div><img src = "http://partsregistry.org/wiki/images/thumb/9/98/CI-5-2.png/800px-CI-5-2.png" width="820"></div>
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<br><b> Figure5-2. </b> Fluorescence of sample at 37℃, 0hr &9 hour.<br><br>
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<br><b> Figure5-2. </b> Fluorescence of sample at 37℃, 0hr &9 hour. X-axis is related green fluorescence intensity, and Y-axis is cells number.<br><br>
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<div><img src = "http://partsregistry.org/wiki/images/thumb/9/95/CI-5-3.png/800px-CI-5-3.png" width="820"></div>
<div><img src = "http://partsregistry.org/wiki/images/thumb/9/95/CI-5-3.png/800px-CI-5-3.png" width="820"></div>
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<br><b> Figure5-3. </b> Fluorescence of sample at 42℃, 0hr & 9 hour.
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<br><b> Figure5-3. </b> Fluorescence of sample at 42℃, 0hr & 9 hour. X-axis is related green fluorescence intensity, and Y-axis is cells number.
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</p>
</p>
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<h2>Discussion</h2>
<h2>Discussion</h2>
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<p>Form the experimental data, we find that MEFL increases steeply at 42℃ in first two hours, which means the expression of GFP is high. Although after the first two hours its performance decreases and maintains at certain volume, it still expresses a lot more than the other two samples in different temperatures. We can regard GFP as our target protein. This means that our hypothesis has been confirmed that any protein placed after this circuit (<a href=http://partsregistry.org/Part: BBa_K098995”>BBa_K098995</a>) can be regulated by elevating or lowering temperature. At higher temperature(42℃), our target protein will perform significantly. On the contrary, at lower temperature(25℃), it will express slightly.</p>
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<p>Form the experimental data, we find that MEFL increases steeply at 42℃ in first two hours, which means the expression of GFP is high. Although after the first two hours its performance decreases and maintains at certain volume, it still expresses a lot more than the other two samples in different temperatures. We can regard GFP as our target protein. This means that our hypothesis has been confirmed that any protein placed after this circuit (<a href=" http://partsregistry.org/Part:BBa_K098995">BBa_K098995</a>) can be regulated by elevating or lowering temperature. At higher temperature(42℃), our target protein will perform significantly. On the contrary, at lower temperature(25℃), it will express slightly.</p>
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Latest revision as of 13:47, 5 October 2011



High Temperature Induced System – cI Promoter & cI repressor

Data

To verify that this circuit can work as expectation, we use GFP (Green Fluorescence Protein, BBa_E0040) as reporter protein. Part BBa_K098988 is also designed by Team Harvard 2008. In this circuit BBa_K098988, we can regard GFP (Green Fluorescence Protein, BBa_E0040) as reporter protein to tell us that the gene after previous circuit BBa_K098995 can be induced or not(Figure 3).



Figure 3. Part BBa_K098988 Design, with Part BBa_K098995 followed by Part BBa_E0240 (include BBa_B0032+BBa_E0040+BBa_B0010+BBa_B0012). Among this circuit, GFP (Green Fluorescence Protein, BBa_E0040) is reporter protein to tell us that the gene after the part BBa_K098995 will be induced or not.

First day, we incubated E.coli which contains this circuit (BBa_K098988) with GFP (Green Fluorescence Protein, BBa_E0040) at 37℃ overnight. The next day, we transferred them to new three tubes with M9 medium. When the O.D.(optical density) reached 0.08, we incubated them to 25℃, 37℃, and 42℃, respectively. We collected the samples once an hour, diluting them 200x for accurate GFP (Green Fluorescence Protein, BBa_E0040) measurements. Here is our experiment original data (Table 1). The data after unit-conversion is shown in Table 2. We can use Table2 data to draw line chart, which is easy for us to compare the mean fluorescence at different temperatures, 25℃, 37℃, and 42℃. (Figure4) The vertical axis is mean fluorescence, and the horizontal axis is time (unit: an hour).

Original Data (Related fluorescent unit)

25℃37℃42℃
09.028.769.14
14.668.3617.54
22.826.4629.24
32.375.0026.44
41.704.6922.27
51.804.4021.94
61.524.7221.57
71.584.7821.01
91.223.922.21

Table 1. Related fluorescent unit at 25℃,37℃,42℃ in 9 hours

Data after unit conversion

25℃37℃42℃
02400.95002326.23502435.4930
11175.90402211.64104926.7290
2683.27851673.73708559.5680
3566.22941268.90407677.1730
4395.39011184.08806377.2450
5420.58761105.15406160.8630
6350.34071192.27706160.8630
7365.31161208.66705988.1660
9276.2400970.06046358.6760

Table 2. Molecules of equivalent fluorescence at 25℃,37℃,42℃ in 9 hours



Figure 4.
Line chart of molecules of equivalent fluorescence performance at 25℃,37℃,42℃ in 9 hours. The vertical axis is mean fluorescence, and the horizontal axis is time. Form the experimental data, we find that MEFL increases steeply at 42℃ in first two hours, which means the expression of GFP is high. Although after the first two hours its performance decreases and maintains at certain volume, it still expresses a lot more than the other two samples at 25℃,37℃.

The following diagrams, Figure5-1.~Figure5-3., are from the Flow cytometer experiment we did. The green fluorescence intensity of E.coli incubated at 25℃ (Figure5-1) and 37℃(Figure5-2) for 9 hours is lower than incubated at 42℃(Figure5-3),which also can refer to the data from Table 1. In Figure5-2., the peak of green fluorescence intensity of E.coli incubated at 37℃ for 9 hours moves right a little bit than incubated at 25℃ for 9 hours. Last, in Figure5-3., incubated at 42℃ for 9 hours, the peak of green fluorescence intensity is significantly stronger than incubated at 25℃ and 37℃ for 9 hours.



Figure5-1. Fluorescence of sample at 25℃, 0hr &9 hour. X-axis is related green fluorescence intensity, and Y-axis is cells number.



Figure5-2. Fluorescence of sample at 37℃, 0hr &9 hour. X-axis is related green fluorescence intensity, and Y-axis is cells number.



Figure5-3. Fluorescence of sample at 42℃, 0hr & 9 hour. X-axis is related green fluorescence intensity, and Y-axis is cells number.


Discussion

Form the experimental data, we find that MEFL increases steeply at 42℃ in first two hours, which means the expression of GFP is high. Although after the first two hours its performance decreases and maintains at certain volume, it still expresses a lot more than the other two samples in different temperatures. We can regard GFP as our target protein. This means that our hypothesis has been confirmed that any protein placed after this circuit (BBa_K098995) can be regulated by elevating or lowering temperature. At higher temperature(42℃), our target protein will perform significantly. On the contrary, at lower temperature(25℃), it will express slightly.