Team:Tokyo Tech/Experiment/Prhl reporter assay

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<li class="current_page_item"><a href="#">Experiment</a>
<li class="current_page_item"><a href="#">Experiment</a>
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        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Experiment/C4HSL-dependent_3oxoC12HSL_production" style="width:400px; margin-left:-135px;">C4HSL-dependent 3oxoC12HSL production</a></li>
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        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Experiment/Plux and Prhl reporter assay" style="width:400px; margin-left:-135px;">Plux and Prhl reporter assay</a></li>
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        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Experiment/3oxoC12HSL-dependent_C4HSL_production" style="width:400px; margin-left:-135px;">3oxoC12HSL-dependent C4HSL production</a></li>
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        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Experiment/Prhl_reporter_assay" style="width:400px; margin-left:-135px;">Improved Prhl reporter assay</a></li>
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        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Experiment/Symbiosis_confirmation_by_co-culture" style="width:400px; margin-left:-135px;">Symbiosis confirmation by co-culture </a></li>
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        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Experiment/C4HSL-dependent_3OC12HSL_production" style="width:400px; margin-left:-135px;">C4HSL-dependent 3OC12HSL production</a></li>
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        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Experiment/Prhl_reporter_assay" style="width:400px; margin-left:-135px;">Prhl reporter assay </a></li>
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        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Experiment/3OC12HSL-dependent_C4HSL_production" style="width:400px; margin-left:-135px;">3OC12HSL-dependent C4HSL production</a></li>
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        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Experiment/Symbiosis_confirmation_by_co-culture" style="width:400px; margin-left:-135px;">Mutualism Confirmation ~Co-culture Assay~</a></li>
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  </ul>
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<li><a href="#">Modeling</a>
<li><a href="#">Modeling</a>
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                                <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Modeling/Modeling1" style="width:260px; margin-left:-63px;">なぞなぞ</a></li>
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                              <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Modeling/Overview" style="width:400px; margin-left:-135px;">Overview</a></li>
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                                <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Modeling/Modeling2" style="width:260px; margin-left:-63px;">Parameter sensitivity analysis</a></li>
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                              <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Modeling/Growth Conditions For Company And Customer" style="width:400px; margin-left:-135px;">Growth Conditions For Company And Customer</a></li>
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                                <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Modeling/Modeling3" style="width:260px; margin-left:-63px;">The effect of economic wave</a></li>
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                              <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Modeling/Analysis of C4HSL-dependent Switch" style="width:400px; margin-left:-135px;">Analysis of C4HSL-dependent Switch</a></li>
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                            </ul>
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                              <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Modeling/Economic Wave"  style="width:400px; margin-left:-135px;">Economic Wave</a></li>
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                          </ul>
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<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Parts">Parts</a></li>
<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Parts">Parts</a></li>
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<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Policy_and_Practices" style="height:45px; padding-top:4px;">Policy and Practices</a></li>
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<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Policy_and_Practices" style="height:50px; padding-top:3px;">Policy and Practices</a></li>
<li><a href="#">More</a>
<li><a href="#">More</a>
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        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Judging_Form" style="width:240px; margin-left:-53px;">Judging Form</a></li>
        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Judging_Form" style="width:240px; margin-left:-53px;">Judging Form</a></li>
        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Sponsors" style="width:240px; margin-left:-53px;">Sponsors</a></li>
        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Sponsors" style="width:240px; margin-left:-53px;">Sponsors</a></li>
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        <li><a href="https://igem.org/Team.cgi" style="width:240px; margin-left:-53px;">Official Team Page</a></li>
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        <li><a href="https://igem.org/Team.cgi?id=1529" style="width:240px; margin-left:-53px;">Official Team Page</a></li>
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<h2 class="title">Experiment</h2>
<h2 class="title">Experiment</h2>
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                 <span class="meta">Prhl reporter assay</span>
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                 <span class="meta">Improved Prhl reporter assay</span>
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                   <td colspan="2"><div align="center" class="title-small">Prhl reporter assay</div></td>
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                    <p class="title-small">Contents </p>
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                      <p class="info-24"><a href="#1">1. Summary of the Experiment</a></p>
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                      <p class="info-24"><a href="#2">2. Results</a></p>
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                      <p class="info-24"><a href="#3">3. Replication </a></p>
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                      <p class="info-24"><a href="#4">4. Materials and Methods </a></p>
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                      <p class="info-18"><a href="#4-1">4-1. Construction</a></p>
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                      <p class="info-18"><a href="#4-2">4-2. Assay Protocol</a></p>
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                      <p class="info-24"><a href="#5">5. Reference </a></p>
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                   <td colspan="2"><h1>1. Summary of the experiment </h1></td>
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                   <td colspan="2"><p class="info-18">Rhl promoter (Prhl) is a  regulatory part activated by RhlR in the presence of N-butyryl-homoserine  lactone (also known as C4-HSL). Existing Rhl promoter (BBa_R0071) has a low  expression level even when it is activated. </p></td>
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                   <td colspan="2"><p class="info-18">                   In order to improve this  expression level, we designed a new Lux promoter which has two RhlR binding  sites instead of two LuxR binding sites (PPrhl_RR: BBa_K1529320). </p></td>
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                   <td colspan="2"><h2>1. Summary of the experiment </h2></td>
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                   <td colspan="2"><p class="info-18">To evaluate the function of this promoter, we  constructed Prhl_RR-GFP plasmids and measured the fluorescence intensity by flow cytometer. In the measurement, we confirmed that GFP under the control of Prhl_RR  showed about ???-folds higher in the fluorescence than that of the original Prhl  (BBa_R0071) (See results).</p></td>
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                   <td colspan="2" class="info-18"><p class="info-18">We added three improved C4HSL-dependent promoters with high maximum expression level by combinations of regulatory-protein binding sites. (Fig. 3-2-1-1.)
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                   <td colspan="2"><p class="info-18">However, our Prhl_RR showed significant leak in the  absence of C4HSL(See results). In order to lessen the leak and increase the  maximum expression level, we newly designed two promoters, Prhl_LR (BBa_K1529310) and Prhl_RL (BBa_K1529300). These promoters have one LuxR binding site and one  RhlR binding site. We changed either the upper RhlR binding site of Prhl_RR to  Lux binding site (Prhl_LR) or the latter RhlR binding site to Lux binding site  (Prhl_RL).</p></td>
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                   <td colspan="2"><p class="info-18">First, we designed an improved Lux promoter which has two RhlR binding sites instead of two LuxR binding sites (Prhl(RR):<a href="http://parts.igem.org/Part:BBa_K1529320"> BBa_K1529320</a>) , as tried in a previous paper (Chuang 2009). To evaluate the function of this promoter, we constructed Prhl(RR)-GFP (<a href="http://parts.igem.org/Part:BBa_K1529321">BBa_K1529321</a>) plasmids and measured the fluorescence intensity by flow cytometer. In the measurement, we confirmed that GFP under the control of Prhl(RR) promoter showed about 20-fold higher in the fluorescence than that of the native Prhl promoter (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>). </p>
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                   <td colspan="2"><p class="info-18">Then, we inserted these promoters to the upstream  of GFP coding sequence and measured the fluorescence intensity. Prhl_LR showed  higher maximum expression level, but also showed significant leak like Prhl_RR.  On the other hand, Prhl_RL had less leak while keeping the high expression  level (See results).</p></td>
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                   <td colspan="2"><p class="info-18">However, Prhl(RR) promoter showed a significant leak in the absence of C4HSL. High level of leak is not suitable for the Company-Customer relationship because their mutualism will be broken. </p>
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                  <td colspan="2"><p class="info-18">In order to lessen the leak and increase the maximum expression level, we newly designed two promoters, Prhl(LR) (<a href="http://parts.igem.org/Part:BBa_K1529310">BBa_K1529310</a>) and Prhl(RL) (<a href="http://parts.igem.org/Part:BBa_K1529300">BBa_K1529300</a>). These promoters have one LuxR binding site and one RhlR binding site. We changed either the former RhlR binding site of Prhl(RR) promoter to LuxR binding site (Prhl(LR)) or the latter RhlR binding site to Lux binding site (Prhl(RL)). </p>
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                  <td colspan="2"><p class="info-18">One of our new promoter, Prhl(RL) improved in its expression level while keeping the low leak. GFP under the control of Prhl(RL) promoter showed about 7-fold higher in the fluorescence than that of the native Prhl promoter. The leak was no more than 2-fold high. </p>
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                  <td colspan="2"><p class="info-18">Although the other Prhl(LR) promoter showed a higher maximum expression level, it showed a significant leak like Prhl(RR) promoter. GFP under the control of Prhl(LR) promoter showed about 7-fold higher in the fluorescence than that of the original Prhl promoter. However, the leak showed no less than 25-fold high. Thus we used our improved Prhl(RL) (<a href="http://parts.igem.org/Part:BBa_K1529300">BBa_K1529300</a>) in the following experiments and modelings. </p>
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                   <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-1.png"><img src="https://static.igem.org/mediawiki/2014/thumb/d/d5/Tokyo_Tech_Fig._3-4-1.png/800px-Tokyo_Tech_Fig._3-4-1.png" alt="" width="450" /></a></div></td>
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                   <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Designs_of_tmproved_Prhl.jpg"><img src="https://static.igem.org/mediawiki/2014/thumb/2/2d/Tokyo_Tech_Designs_of_tmproved_Prhl.jpg/800px-Tokyo_Tech_Designs_of_tmproved_Prhl.jpg" alt="" width="650" /></a></div></td>
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                   <td colspan="2"><div align="center">Fig. 3-4-1. The design of our Rhl promoters</div></td>
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                   <td colspan="2"><div align="center"><strong>Fig. 3-2-1-1.</strong> The design of our Prhl promoters</div></td>
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                   <td colspan="2"><h1>2. Results</h1></td>
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                   <td colspan="2"><p class="info-18">We measured GFP expression with the four different promoters (Prhl (BBa_R0071), Prhl_RR (BBa_K1529320), Prhl_LR (BBa_K1529310) and Prhl_RL (BBa_K1529300)) by flow cytometer. Each promoter was tested in the presence and also in the absence of C4HSL (See Materials and Methods for detailed procedures).</p></td>
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                  <td colspan="2"><h2>2. Results</h2></td>
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                   <td colspan="2"><p class="info-18">We measured the GFP expression with the four different promoters (Prhl (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>), Prhl(RR) (<a href="http://parts.igem.org/Part:BBa_K1529320">BBa_K1529320</a>), Prhl(LR) (<a href="http://parts.igem.org/Part:BBa_K1529310">BBa_K1529310</a>), and Prhl(RL) (<a href="http://parts.igem.org/Part:BBa_K1529300">BBa_K1529300</a>)) by flow cytometer(Fig. 3-2-2-1). Each promoter was tested in the presence and also in the absence of C4HSL (See Materials and Methods for detailed procedures).</p>
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                   <td colspan="2"><div align="center">Fig. 3-4-2. The four promoters we tested</div></td>
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                   <td colspan="2"><div align="center"><strong>Fig. 3-2-2-1.</strong> The four promoters we have tested</div></td>
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                   <td colspan="2"><p class="info-18">Fig. 3-4-3 shows the fluorescence intensity detected by flow cytometer. Fig. 3-4-4 is the extracted data which shows the comparison of Prhl, Prhl_RR, and Prhl_RL</p></td>
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                   <td colspan="2"><p class="info-18">Fig. 3-2-2-2 shows the fluorescence intensity detected by flow cytometer. Fig. 3-2-2-3 is the extracted data which shows the comparison of the three promoters: Prhl, Prhl(RR), and Prhl(RL).</p>
 +
                    <div>
 +
                      <div class="info-18"></div>
 +
                    </div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><p class="info-18">As Fig 3-4-4 shows, when C4HSL is induced, Prhl_RR showed higher maximum expression level and higher leak than the original Prhl.</p>                  </td>
+
                   <td colspan="2"><p class="info-18">As Fig. 3-2-2-2 shows, when induced by C4HSL, Prhl(RR) promoter showed higher maximum expression level and higher leak than the native Prhl promoter. </p>
 +
                    <div>
 +
                      <div> </div>
 +
                      <div> </div>
 +
                  </div>                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2"><p class="info-18">Although Prhl(RL) promoter had lower maximum expression level compared to Prhl(RR) promoter, it had the highest induced/not-induced ratio. This means Prhl(RL) promoter has little leak. Therefore, we can say that Prhl(RL) promoter is the best improved Prhl promoter due to the advantages of less leak and higher expression level. </p>
 +
                    <div>
 +
                      <div> </div>
 +
                      <div> </div>
 +
                      <div> </div>
 +
                      <div> </div>
 +
                      <div> </div>
 +
                    </div>
 +
<div>
 +
                  <div> </div>
 +
                      <div> </div>
 +
                      <div> </div>
 +
                      <div> </div>
 +
                      <div> </div>
 +
                  </div></td>
                 </tr>
                 </tr>
                 <tr>
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                   <td><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-3.png"><img src="https://static.igem.org/mediawiki/2014/thumb/2/26/Tokyo_Tech_Fig._3-4-3.png/800px-Tokyo_Tech_Fig._3-4-3.png" alt="" width="400" /></a></td>
+
                   <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-3.png"><img src="https://static.igem.org/mediawiki/2014/thumb/2/26/Tokyo_Tech_Fig._3-4-3.png/800px-Tokyo_Tech_Fig._3-4-3.png" alt="" width="750" /></a></div></td>            
-
                  <td><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-4.png"><img src="https://static.igem.org/mediawiki/2014/thumb/6/6e/Tokyo_Tech_Fig._3-4-4.png/800px-Tokyo_Tech_Fig._3-4-4.png" alt="" width="400" /></a></td>
+
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><div align="center">Fig. 3-4-3. The Fluorescence intensity of the cells <br />
+
                   <td colspan="2"><div align="center"><strong>Fig. 3-2-2-2.</strong> The fluorescence intensity of the cells (with positive and negative controls)</div></td>
-
                  (with positive and negative controls)</div></td>
+
-
                  <td><div align="center">Fig. 3-4-4. The fluorescence intensity of the cells  with original Prhl (BBa_R0071), Prhl_RR (BBa_K1529320), Prhl_RL (BBa_K1529300)</div></td>
+
                 </tr>
                 </tr>
                 <tr>
                 <tr>
                   <td>&nbsp;</td>
                   <td>&nbsp;</td>
                   <td>&nbsp;</td>
                   <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-4.png"><img src="https://static.igem.org/mediawiki/2014/6/6e/Tokyo_Tech_Fig._3-4-4.png" alt="" width="400" /></a></div></td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2"><div align="center"><strong>Fig. 3-2-2-3.</strong> The fluorescence intensity of the cells with the native Prhl (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>), Prhl(RR) (<a href="http://parts.igem.org/Part:BBa_K1529320">BBa_K1529320</a>), and Prhl(RL) (<a href="http://parts.igem.org/Part:BBa_K1529300">BBa_K1529300</a>) promoter</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2">&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2" class="entry-long"><a name="3" id="3"></a></td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2">&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2"><h2>3. Replication</h2></td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2"><p class="info-18">To confirm the intensity of Prhl(RL) promoter, we did replication study of the reporter assay.</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2"><p class="info-18">We prepared three plasmids shown below and measured the fluorescence intensity by GFP expression when we added the signaling molecules. Detail of the protocol of this study is written in the <a href="https://2014.igem.org/Team:Tokyo_Tech/Experiment/Plux_and_Prhl_reporter_assay">Plux and Prhl promoter assay page.</a></p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2"><p class="info-18">- Ptet-LuxR(6A1), Plux-GFP(3K3)</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2"><p class="info-18">- Ptet-RhlR(6A1), Prhl-GFP(3K3)</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2"><p class="info-18">- Ptet-RhlR(6A1), Prhl(RL)-GFP(3K3)</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Plux_Prhl_Reporter_Assay_Replication_Result.png"><img src="https://static.igem.org/mediawiki/2014/2/2f/Tokyo_Tech_Plux_Prhl_Reporter_Assay_Replication_Result.png" width="500"/></a></div></td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2"><div align="center"><strong>Fig. 3-2-3-1.</strong> The fluorescence intensity of the cells with Plux, Prhl and Prhl(RL) promoter.</div></td>
 +
                </tr>
 +
                <tr>
 +
                <td colspan="2"><p class="info-18">Fig. 3-2-3-1 shows that Prhl(RL) promoter is still weaker than Plux promoter. Prhl(RL) promoter does not have enough power for our project, so we have to improve Prhl promoter further.</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2">&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2" class="entry-long"><span class="entry-long" style="clear: both;"><a name="4" id="4"></a></span></td>
                 </tr>
                 </tr>
                 <tr>
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                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><h1>3. Materials and methods</h1></td>
+
                   <td colspan="2"><h2>4. Materials and methods <a name="4-1" id="4-1"></a></h2></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2" class="head">3-1. Construction</td>
+
                   <td colspan="2" class="head">4-1. Construction</td>
                 </tr>
                 </tr>
                 <tr>
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                   <td colspan="2"><div align="center">
                   <td colspan="2"><div align="center">
                     <blockquote>
                     <blockquote>
-
                       <p><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-5.png"><img src="https://static.igem.org/mediawiki/2014/2/27/Tokyo_Tech_Fig._3-4-5.png" alt="" width="800" /></a></p>
+
                       <p><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-5.png"><img src="https://static.igem.org/mediawiki/2014/2/27/Tokyo_Tech_Fig._3-4-5.png" alt="" width="500" /></a></p>
                     </blockquote>
                     </blockquote>
                   </div></td>
                   </div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><div align="center">Fig. 3-4-5 </div></td>
+
                   <td colspan="2"><div align="center"><strong>Fig. 3-2-4-1.</strong> </div></td>
                 </tr>
                 </tr>
                 <tr>
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                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><p class="info-18">     B. Ptet-RhlR (pSB6A1)　Prhl_RR-GFP (pSB3K3)</p></td>
+
                   <td colspan="2"><p class="info-18">B. Ptet-RhlR (pSB6A1)　Prhl(RR)-GFP (pSB3K3)</p></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
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                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-6.png"><img src="https://static.igem.org/mediawiki/2014/1/10/Tokyo_Tech_Fig._3-4-6.png" alt="" width="800" /></a></div></td>
+
                   <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-6.png"><img src="https://static.igem.org/mediawiki/2014/1/10/Tokyo_Tech_Fig._3-4-6.png" alt="" width="500" /></a></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><div align="center">Fig. 3-4-6 </div></td>
+
                   <td colspan="2"><div align="center"><strong>Fig. 3-2-4-2.</strong> </div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
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                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><p class="info-18">    C. Ptet-RhlR (pSB6A1)　Prhl_LR-GFP (pSB3K3)</p></td>
+
                   <td colspan="2"><p class="info-18">    C. Ptet-RhlR (pSB6A1)　Prhl(LR)GFP (pSB3K3)</p></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
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                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-7.png"><img src="https://static.igem.org/mediawiki/2014/5/50/Tokyo_Tech_Fig._3-4-7.png" alt="" width="800" /></div></td>
+
                   <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-7.png"><img src="https://static.igem.org/mediawiki/2014/5/50/Tokyo_Tech_Fig._3-4-7.png" alt="" width="500" /></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><div align="center">Fig. 3-4-7</div></td>
+
                   <td colspan="2"><div align="center"><strong>Fig. 3-2-4-3.</strong></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
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                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><p class="info-18">    D. Ptet-RhlR (pSB6A1)　Prhl_RL-GFP (pSB3K3)</p></td>
+
                   <td colspan="2"><p class="info-18">    D. Ptet-RhlR (pSB6A1)　Prhl(RL)-GFP (pSB3K3)</p></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
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                 </tr>
                 </tr>
                 <tr>
                 <tr>
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                   <td colspan="2"><div align="center"><a href"https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-8.png"><img src="https://static.igem.org/mediawiki/2014/9/98/Tokyo_Tech_Fig._3-4-8.png" alt="" width="800" /></a></div></td>
+
                   <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-8.png"><img src="https://static.igem.org/mediawiki/2014/9/98/Tokyo_Tech_Fig._3-4-8.png" alt="" width="500" /></a></div></td>
                 </tr>
                 </tr>
 +
                <tr>
 +
                  <td colspan="2"><div align="center"><strong>Fig. 3-2-4-4.</strong></div></td>
 +
                </tr>               
                 <tr>
                 <tr>
                   <td colspan="2">&nbsp;</td>
                   <td colspan="2">&nbsp;</td>
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                 </tr>
                 </tr>
                 <tr>
                 <tr>
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                   <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-9.png"><img src="https://static.igem.org/mediawiki/2014/1/1a/Tokyo_Tech_Fig._3-4-9.png" alt="" width="800" /></a></div></td>
+
                   <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-9.png"><img src="https://static.igem.org/mediawiki/2014/1/1a/Tokyo_Tech_Fig._3-4-9.png" alt="" width="500" /></a></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><div align="center">Fig. 3-4-9</div></td>
+
                   <td colspan="2"><div align="center"><strong>Fig. 3-2-4-5.</strong></div></td>
                 </tr>
                 </tr>
                 <tr>
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                 </tr>
                 </tr>
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-
                   <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-10.png"><img src="https://static.igem.org/mediawiki/2014/0/0a/Tokyo_Tech_Fig._3-4-10.png" alt="" width="800" /></a></div></td>
+
                   <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-10.png"><img src="https://static.igem.org/mediawiki/2014/0/0a/Tokyo_Tech_Fig._3-4-10.png" alt="" width="500" /></a></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><div align="center">Fig. 3-4-10</div></td>
+
                   <td colspan="2"><div align="center"><strong>Fig. 3-2-4-6.</strong></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2">&nbsp;</td>
+
                   <td colspan="2">&nbsp;<a name="3-2" id="3-2"></a></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2" class="head">3-2. Assay Protocol</td>
+
                   <td colspan="2" class="head">4-2. Assay Protocol</td>
                 </tr>
                 </tr>
                 <tr>
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                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><p class="info-18">1. Prepare  2 overnight cultures for each samples A~F in 3 mL LB medium, containing  ampicillin (50 microg /mL) and kanamycin (30 microg / mL) at 37°C for 12 h.</p></td>
+
                   <td colspan="2" class="info-18">1. Prepare  2 overnight cultures for each sample A~F in 3 mL LB medium, containing  ampicillin (50 microg /mL) and kanamycin (30 microg / mL) at 37°C for 12 h.</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><p class="info-18">2. Dilute the overnight cultures to 1 / 100 in fresh LB medium (3 mL)  containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) (→fresh  culture). Make glycerol stocks from the remainders.</p>                    </td>
+
                   <td colspan="2" class="info-18">2. Dilute the overnight cultures to 1 / 100 in fresh LB medium (3 mL)  containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) (→fresh  culture).                     </td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><p class="info-18">3. Incubate the fresh cultures in 37°C until the observed OD590  reaches 0.3 (Actual value 0.42).</p></td>
+
                   <td colspan="2" class="info-18">3. Incubate the fresh cultures in 37°C until the OD590  reaches 0.3.</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><p class="info-18">4. Add 30 microL of 500 microM C4HSL or DMSO as listed below:</p>                  </td>
+
                   <td colspan="2" class="info-18">4. Add 30 microL of 500 microM C4HSL or DMSO as listed below:             </td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><blockquote class="info-18">                     A-5 μM: A + C4HSL</blockquote></td>
+
                   <td colspan="2" class="info-18" style="text-indent:50px;"> A-5 microM: A + C4HSL</blockquote></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><blockquote class="info-18">                     A-0 μM: A + DMSO</blockquote></td>
+
                   <td colspan="2" class="info-18" style="text-indent:50px;">A-0 microM: A + DMSO</blockquote></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><blockquote class="info-18">B-5 μM: B + C4HSL</blockquote></td>
+
                   <td colspan="2" class="info-18" style="text-indent:50px;">B-5 microM: B + C4HSL</blockquote></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><blockquote class="info-18">B-0 μM: B + DMSO</blockquote></td>
+
                   <td colspan="2" class="info-18" style="text-indent:50px;">B-0 microM: B + DMSO</blockquote></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><blockquote class="info-18">C-5 μM: C + C4HSL</blockquote></td>
+
                   <td colspan="2" class="info-18" style="text-indent:50px;">C-5 microM: C + C4HSL</blockquote></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><blockquote class="info-18">C-0 μM: C + DMSO</blockquote></td>
+
                   <td colspan="2" class="info-18" style="text-indent:50px;">C-0 microM: C + DMSO</blockquote></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><blockquote class="info-18">D-5 μM: D + C4HSL</blockquote></td>
+
                   <td colspan="2" class="info-18" style="text-indent:50px;">D-5 microM: D + C4HSL</blockquote></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><blockquote class="info-18">D-0 μM: D + DMSO</blockquote></td>
+
                   <td colspan="2" class="info-18" style="text-indent:50px;">D-0 microM: D + DMSO</blockquote></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                  <td colspan="2"><blockquote class="info-18">E-5  μM: E + C4HSL</blockquote></td>
+
                  <td colspan="2" class="info-18" style="text-indent:50px;">E-5 microM: E + C4HSL</blockquote></td>
 +
                </tr>
 +
                <td colspan="2" class="info-18" style="text-indent:50px;">E-0 microM: E + DMSO</blockquote></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><blockquote class="info-18">E-0  μM: E + DMSO</blockquote></td>
+
                   <td colspan="2" class="info-18" style="text-indent:50px;">F-5 microM: F + C4HSL</blockquote></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><blockquote class="info-18">F-5  μM: F + C4HSL</blockquote></td>
+
                   <td colspan="2" class="info-18" style="text-indent:50px;">F-0 microM: F + DMSO</blockquote></td>
-
                 </tr>
+
                 </tr>  <td colspan="2"><td>
                 <tr>
                 <tr>
-
                   <td colspan="2"><blockquote class="info-18">F-0  μM: F + DMSO</blockquote></td>
+
                   <td colspan="2" class="info-18">5. Incubate the samples at 37°C for 4 h. </p></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><p class="info-18">5. Incubate the samples at 37°C for 4 h.</p></td>
+
                   <td colspan="2" class="info-18">6. Start preparing the flow cytometer 1 h before the end of incubation.</p>                   </td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><p class="info-18">6. Start preparing the flow cytometer 1 h before the end of incubation.</p></td>
+
                   <td colspan="2" class="info-18">7. Take 200 microL of the sample, and centrifuge at 9000x g, 1 min., 4°C.</p>                   </td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><p class="info-18">7. Take 200 microL of the sample, and centrifuge at 9000 Xg, 1 min,  4°C.</p></td>
+
                   <td colspan="2" class="info-18">8. Remove the supernatant by using P1000 pipette.</p>                   </td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><p class="info-18">8. Remove the supernatant by using P1000 pipette.</p></td>
+
                   <td colspan="2" class="info-18">9. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend.</p>                   </td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><p class="info-18">9. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend.</p></td>
+
                   <td colspan="2" class="info-18">10.Dispense all of each suspension into a disposable tube through a cell strainer. </p>                   </td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><p class="info-18">10. Dispense all of each suspension into a disposable tube through a  cell strainer. </p></td>
+
                   <td colspan="2" class="info-18">11.Measure fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).</p>                   </td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td colspan="2"><p class="info-18">11. Measure fluorescence intensity with a flow cytometer (We used BD  FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).</p></td>
+
                   <td colspan="2">&nbsp;</td>
                 </tr>
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                   <td colspan="2">&nbsp;</td>
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                   <td colspan="2" class="entry-long">&nbsp;<a name="5" id="5"></a></td>
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                   <td colspan="2"><h1>4. References</h1></td>
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                   <td colspan="2"><h2>5. Reference</h2></td>
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                   <td colspan="2"><p class="info-18">[1] “Simpson’s Paradox in a Synthetic Microbial System” John S. Chuang, Olivier Rivoire, Stanislas Leibler 9 JANUARY  2009 VOL 323 SCIENCE</p></td>
+
                   <td colspan="2"><p class="info-18">John S. Chuang et al. (2009) Simpson’s Paradox in a Synthetic Microbial System. SCIENCE 323: 272-275</p>
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              <p>&nbsp;</p>
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Latest revision as of 03:34, 18 October 2014

Tokyo_Tech

Experiment

Improved Prhl reporter assay

Contents

  1. 1. Summary of the Experiment

  3. 2. Results

  4. 3. Replication

  5. 4. Materials and Methods

  6. 4-1. Construction

  7. 4-2. Assay Protocol

  8. 5. Reference

 
 
 

1. Summary of the experiment

We added three improved C4HSL-dependent promoters with high maximum expression level by combinations of regulatory-protein binding sites. (Fig. 3-2-1-1.)

First, we designed an improved Lux promoter which has two RhlR binding sites instead of two LuxR binding sites (Prhl(RR): BBa_K1529320) , as tried in a previous paper (Chuang 2009). To evaluate the function of this promoter, we constructed Prhl(RR)-GFP (BBa_K1529321) plasmids and measured the fluorescence intensity by flow cytometer. In the measurement, we confirmed that GFP under the control of Prhl(RR) promoter showed about 20-fold higher in the fluorescence than that of the native Prhl promoter (BBa_R0071).

However, Prhl(RR) promoter showed a significant leak in the absence of C4HSL. High level of leak is not suitable for the Company-Customer relationship because their mutualism will be broken.

In order to lessen the leak and increase the maximum expression level, we newly designed two promoters, Prhl(LR) (BBa_K1529310) and Prhl(RL) (BBa_K1529300). These promoters have one LuxR binding site and one RhlR binding site. We changed either the former RhlR binding site of Prhl(RR) promoter to LuxR binding site (Prhl(LR)) or the latter RhlR binding site to Lux binding site (Prhl(RL)).

One of our new promoter, Prhl(RL) improved in its expression level while keeping the low leak. GFP under the control of Prhl(RL) promoter showed about 7-fold higher in the fluorescence than that of the native Prhl promoter. The leak was no more than 2-fold high.

Although the other Prhl(LR) promoter showed a higher maximum expression level, it showed a significant leak like Prhl(RR) promoter. GFP under the control of Prhl(LR) promoter showed about 7-fold higher in the fluorescence than that of the original Prhl promoter. However, the leak showed no less than 25-fold high. Thus we used our improved Prhl(RL) (BBa_K1529300) in the following experiments and modelings.

 
Fig. 3-2-1-1. The design of our Prhl promoters
 
 
 

2. Results

We measured the GFP expression with the four different promoters (Prhl (BBa_R0071), Prhl(RR) (BBa_K1529320), Prhl(LR) (BBa_K1529310), and Prhl(RL) (BBa_K1529300)) by flow cytometer(Fig. 3-2-2-1). Each promoter was tested in the presence and also in the absence of C4HSL (See Materials and Methods for detailed procedures).
 
Fig. 3-2-2-1. The four promoters we have tested
 

Fig. 3-2-2-2 shows the fluorescence intensity detected by flow cytometer. Fig. 3-2-2-3 is the extracted data which shows the comparison of the three promoters: Prhl, Prhl(RR), and Prhl(RL).

As Fig. 3-2-2-2 shows, when induced by C4HSL, Prhl(RR) promoter showed higher maximum expression level and higher leak than the native Prhl promoter.

Although Prhl(RL) promoter had lower maximum expression level compared to Prhl(RR) promoter, it had the highest induced/not-induced ratio. This means Prhl(RL) promoter has little leak. Therefore, we can say that Prhl(RL) promoter is the best improved Prhl promoter due to the advantages of less leak and higher expression level.

   
Fig. 3-2-2-2. The fluorescence intensity of the cells (with positive and negative controls)
   
   
Fig. 3-2-2-3. The fluorescence intensity of the cells with the native Prhl (BBa_R0071), Prhl(RR) (BBa_K1529320), and Prhl(RL) (BBa_K1529300) promoter
   
 
 

3. Replication

To confirm the intensity of Prhl(RL) promoter, we did replication study of the reporter assay.

We prepared three plasmids shown below and measured the fluorescence intensity by GFP expression when we added the signaling molecules. Detail of the protocol of this study is written in the Plux and Prhl promoter assay page.

- Ptet-LuxR(6A1), Plux-GFP(3K3)

- Ptet-RhlR(6A1), Prhl-GFP(3K3)

- Ptet-RhlR(6A1), Prhl(RL)-GFP(3K3)
   
Fig. 3-2-3-1. The fluorescence intensity of the cells with Plux, Prhl and Prhl(RL) promoter.

Fig. 3-2-3-1 shows that Prhl(RL) promoter is still weaker than Plux promoter. Prhl(RL) promoter does not have enough power for our project, so we have to improve Prhl promoter further.
 
 

4. Materials and methods

4-1. Construction
 
-Strain

All the samples were JM2.300 strain

 
-Plasmids

A. Ptet-RhlR (pSB6A1), Prhl-GFP (pSB3K3)
 

Fig. 3-2-4-1.
 

B. Ptet-RhlR (pSB6A1)　Prhl(RR)-GFP (pSB3K3)
 
Fig. 3-2-4-2.
 

C. Ptet-RhlR (pSB6A1)　Prhl(LR)GFP (pSB3K3)
 
Fig. 3-2-4-3.
 

D. Ptet-RhlR (pSB6A1)　Prhl(RL)-GFP (pSB3K3)
 
Fig. 3-2-4-4.
 

E. Ptet-RhlR (pSB6A1)　PlacUV5-GFP (pSB3K3) ...Positive control
 
Fig. 3-2-4-5.
 

F. Ptet-RhlR (pSB6A1)　promoter less-GFP(pSB3K3) ...Negative control
 
Fig. 3-2-4-6.
 
4-2. Assay Protocol
 
1. Prepare 2 overnight cultures for each sample A~F in 3 mL LB medium, containing ampicillin (50 microg /mL) and kanamycin (30 microg / mL) at 37°C for 12 h.
2. Dilute the overnight cultures to 1 / 100 in fresh LB medium (3 mL) containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) (→fresh culture).
3. Incubate the fresh cultures in 37°C until the OD590 reaches 0.3.
4. Add 30 microL of 500 microM C4HSL or DMSO as listed below:
A-5 microM: A + C4HSL
A-0 microM: A + DMSO
B-5 microM: B + C4HSL
B-0 microM: B + DMSO
C-5 microM: C + C4HSL
C-0 microM: C + DMSO
D-5 microM: D + C4HSL
D-0 microM: D + DMSO
E-5 microM: E + C4HSL
E-0 microM: E + DMSO
F-5 microM: F + C4HSL
F-0 microM: F + DMSO
5. Incubate the samples at 37°C for 4 h.

6. Start preparing the flow cytometer 1 h before the end of incubation.
7. Take 200 microL of the sample, and centrifuge at 9000x g, 1 min., 4°C.
8. Remove the supernatant by using P1000 pipette.
9. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend.
10.Dispense all of each suspension into a disposable tube through a cell strainer.

11.Measure fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).
 
 
 

5. Reference

John S. Chuang et al. (2009) Simpson’s Paradox in a Synthetic Microbial System. SCIENCE 323: 272-275

 

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