Line 476: | Line 476: | ||
<h4><strong>①Amplify</strong> </h4> | <h4><strong>①Amplify</strong> </h4> | ||
<p> | <p> | ||
− | <I><I><I><I><I><I><I>pmrA-B-Fe</I></I></I></I></I></I></I>、 <I> <I> <I> <I> pmrC- | + | <I><I><I><I><I><I><I>pmrA-B-Fe</I></I></I></I></I></I></I>、 <I> <I> <I> <I> pmrC-GFP </I></I></I></I> gene through the pcr protocol |
</p> | </p> | ||
<h4><strong>②Plasmid Construction</strong> </h4> | <h4><strong>②Plasmid Construction</strong> </h4> | ||
<p> | <p> | ||
− | Coding sequences <I> pmrA-pmrB</I> and <I> <I> <I> <I> pmrC- | + | Coding sequences <I> pmrA-pmrB</I> and <I> <I> <I> <I> pmrC-GFP </I></I></I></I>were cloned into the expression vector pBAD30 by restriction enzyme digestion and DNA ligation. |
</p> | </p> | ||
<div class="experiment_circuit col-md-4 col-xs-4 col-md-offset-4 col-xs-offset-4"> | <div class="experiment_circuit col-md-4 col-xs-4 col-md-offset-4 col-xs-offset-4"> | ||
Line 494: | Line 494: | ||
</p> | </p> | ||
<p> | <p> | ||
− | We use the original <I> <I> pmrA-pmrB-Fe </I> </I>-<I> <I> <I> <I> pmrC- | + | We use the original <I> <I> pmrA-pmrB-Fe </I> </I>-<I> <I> <I> <I> pmrC-GFP </I></I></I></I>circuit to test whether our |
pmrABC system works in our plasmid, then the expression and efficacy | pmrABC system works in our plasmid, then the expression and efficacy | ||
of LBT1-12 were tested . | of LBT1-12 were tested . | ||
Line 512: | Line 512: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
− | <td>pmrA-pmrB/Fe- <I> pmrC- | + | <td>pmrA-pmrB/Fe- <I> pmrC-GFP </I></td> |
<td>pBAD30</td> | <td>pBAD30</td> | ||
<td>Test whether our pmrABC system works in our plasmid</td> | <td>Test whether our pmrABC system works in our plasmid</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>pmrA-pmrB/LBT1- <I> <I> <I> <I> pmrC- | + | <td>pmrA-pmrB/LBT1- <I> <I> <I> <I> pmrC-GFP </I></I></I></I></td> |
<td>pBAD30</td> | <td>pBAD30</td> | ||
<td> Detecting effectiveness of protein LBT1</td> | <td> Detecting effectiveness of protein LBT1</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>pmrA-pmrB/LBT2- <I> pmrC- | + | <td>pmrA-pmrB/LBT2- <I> pmrC-GFP </I></td> |
<td>pBAD30 </td> | <td>pBAD30 </td> | ||
<td> Detecting effectiveness of protein LBT2</td> | <td> Detecting effectiveness of protein LBT2</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>pmrA-pmrB/LBT3- <I> pmrC- | + | <td>pmrA-pmrB/LBT3- <I> pmrC-GFP </I></td> |
<td>pBAD30 </td> | <td>pBAD30 </td> | ||
<td>Detecting effectiveness of protein LBT3</td> | <td>Detecting effectiveness of protein LBT3</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>pmrA-pmrB/LBT4- <I> pmrC- | + | <td>pmrA-pmrB/LBT4- <I> pmrC-GFP </I></td> |
<td> pBAD30 </td> | <td> pBAD30 </td> | ||
<td>Detecting effectiveness of protein LBT4</td> | <td>Detecting effectiveness of protein LBT4</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>pmrA-pmrB/LBT5- <I> pmrC- | + | <td>pmrA-pmrB/LBT5- <I> pmrC-GFP </I></td> |
<td>pBAD30</td> | <td>pBAD30</td> | ||
<td>Detecting effectiveness of protein LBT5</td> | <td>Detecting effectiveness of protein LBT5</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>pmrA-pmrB/LBT6- <I> <I> <I> <I> pmrC- | + | <td>pmrA-pmrB/LBT6- <I> <I> <I> <I> pmrC-GFP </I></I></I></I></td> |
<td>pBAD30</td> | <td>pBAD30</td> | ||
<td> Detecting effectiveness of protein LBT6</td> | <td> Detecting effectiveness of protein LBT6</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>pmrA-pmrB/LBT7- <I> pmrC- | + | <td>pmrA-pmrB/LBT7- <I> pmrC-GFP </I></td> |
<td>pBAD30 </td> | <td>pBAD30 </td> | ||
<td> Detecting effectiveness of protein LBT7</td> | <td> Detecting effectiveness of protein LBT7</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>pmrA-pmrB/LBT8- <I> pmrC- | + | <td>pmrA-pmrB/LBT8- <I> pmrC-GFP </I></td> |
<td>pBAD30 </td> | <td>pBAD30 </td> | ||
<td>Detecting effectiveness of protein LBT8</td> | <td>Detecting effectiveness of protein LBT8</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>pmrA-pmrB/LBT9- <I> pmrC- | + | <td>pmrA-pmrB/LBT9- <I> pmrC-GFP </I></td> |
<td> pBAD30 </td> | <td> pBAD30 </td> | ||
<td>Detecting effectiveness of protein LBT9</td> | <td>Detecting effectiveness of protein LBT9</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>pmrA-pmrB/LBT10- <I> pmrC- | + | <td>pmrA-pmrB/LBT10- <I> pmrC-GFP </I></td> |
<td>pBAD30 </td> | <td>pBAD30 </td> | ||
<td> Detecting effectiveness of protein LBT10</td> | <td> Detecting effectiveness of protein LBT10</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>pmrA-pmrB/LBT11- <I> pmrC- | + | <td>pmrA-pmrB/LBT11- <I> pmrC-GFP </I></td> |
<td>pBAD30 </td> | <td>pBAD30 </td> | ||
<td>Detecting effectiveness of protein LBT11</td> | <td>Detecting effectiveness of protein LBT11</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>pmrA-pmrB/LBT12- <I> <I> <I> <I> pmrC- | + | <td>pmrA-pmrB/LBT12- <I> <I> <I> <I> pmrC-GFP </I></I></I></I></td> |
<td> pBAD30 </td> | <td> pBAD30 </td> | ||
<td>Detecting effectiveness of protein LBT12</td> | <td>Detecting effectiveness of protein LBT12</td> | ||
Line 604: | Line 604: | ||
<h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Construction of the whole circuit</h3> | <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Construction of the whole circuit</h3> | ||
<p> | <p> | ||
− | <I> pmrA-pmrB-Fe-pmrC- | + | <I> pmrA-pmrB-Fe-pmrC-GFP</I> All of our part should be built in one plasmid. Therefore, we use endonuclease and ligase to replace the GFP by Oprf-LBT and Oprf-Si tag. |
</p> | </p> | ||
<img title="demo1" src="https://static.igem.org/mediawiki/2017/5/58/2017_HUST_China_Experiment_image4.png" alt="experiment_circuit" alt="demo1" class="col-xs-4 col-xs-offset-4" style="padding: 10px 0px;"> | <img title="demo1" src="https://static.igem.org/mediawiki/2017/5/58/2017_HUST_China_Experiment_image4.png" alt="experiment_circuit" alt="demo1" class="col-xs-4 col-xs-offset-4" style="padding: 10px 0px;"> |
Revision as of 15:43, 31 October 2017