Team:Tokyo Tech/Experiment/3OC12HSL-dependent C4HSL production

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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/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/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/Prhl_reporter_assay" style="width:400px; margin-left:-135px;">Improved Prhl reporter assay</a></li>
        <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>
        <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>
        <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>
        <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;">Symbiosis confirmation by co-culture </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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        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Experiment/Transporter_assay" style="width:400px; margin-left:-135px;">Transporter assay</a></li>
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  </ul>
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<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Modeling">Modeling</a></li>
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<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Modeling">Modeling</a>
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                          <ul>
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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/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/Analysis of C4HSL-dependent Switch" style="width:400px; margin-left:-135px;">Analysis of C4HSL-dependent Switch</a></li>
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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>
<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>
<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="https://2014.igem.org/Team:Tokyo_Tech/Policy_and_Practices" style="height:50px; padding-top:3px;">Policy and Practices</a></li>
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<h2 class="title">Experiment</h2>
<h2 class="title">Experiment</h2>
                 <span class="meta">3OC12HSL-dependent C4HSL production</span>
                 <span class="meta">3OC12HSL-dependent C4HSL production</span>
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               <p>&nbsp;</p>
               <p>&nbsp;</p>
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              <p align="center"><span class="title-small">Contents</span></p>
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          <p align="left" class="info-24"><a href="#Introduction">1. Introduction </a></p>  
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            <p align="left" class="info-24"><a href="#Summary">2. Summary of the Experiments</a></p>
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            <p align="left" class="info-18"><a href="#Summary">2-1. 3OC12HSL-dependent CmR expression</a></p>
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            <p align="left" class="info-18"><a href="#2.2">2-2. 3OC12HSL-dependent C4HSL production</a></p>
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                    <p align="left" class="info-24"><a href="#Results">3. Results </a></p>
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                    <p align="left" class="info-18"><a href="#Results">3.1. 3OC12HSL-dependent CmR expression</a></p>
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<p align="left" class="info-18"><a href="#3.2">3.2. 3OC12HSL-dependent C4HSL production</a></p>
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                    <p align="left" class="info-24"><a href="#Materials">4. Materials and Methods</a></p>
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      <p align="left" class="info-18"><a href="#4.1">4.1. Construction</a></p>
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    <p align="left" class="info-18"><a href="#4.2">4.2. Assay Protocol </a></p>
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    <p align="left" class="info" style="text-indent:40px;"><a href="#4.2.1">4.2.1. 3OC12HSL-depemdent CmR expression</a></p>
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    <p align="left" class="info" style="text-indent:40px;"><a href="#4.2.2">4.2.2. C4HSL-Dependent 3OC12HSL Production Assay</a></p>
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    <p align="left" class="info-24"><a href="#Reference">5. Reference</a></p>
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                <div class="title" style="clear: both;">&nbsp;</div>
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                   <td width="890"><div align="center" class="title-small">3OC12HSL-dependent C4HSL production module</div></td>
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                   <td width="890" class="entry-long"><a name="Introduction" id="Introduction"></a></td>
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                   <td>&nbsp;</td>
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                  <td><h2>1. Introduction</h2></td>
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                   <td><h2>1. Summary of the experiment </h2></td>
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                   <td><p class="info-18">We designed a signal-dependent signal production in our system by using signaling molecules and antibiotics resistance gene. In our bank story, we used signaling molecule 3OC12HSL as product.</p></td>
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                  <td><p class="info-18">For construction of the 3OC12HSL-dependent chloramphenicol resistance gene product(CmR) and C4HSL production module, we constructed a new part Plux-CmR-RhlI (<a href="http://parts.igem.org/Part:BBa_K1529797">BBa_K1529797</a>). Plux-CmR-RhlI cell is an engineered <i>E. coli</i> that contains 3OC12HSL-dependent RhlI generator and a constitutive LuxR generator. As a constitutive LuxR generator, we used Ptet-LuxR. In our bank story, this part imitates the function of Customer. (Fig. 3-4-1-1) We confirmed the 3OC12HSL-dependent growth by measuring the optical density, and 3OC12HSL-dependent C4HSL production by using reporter cell.</p></td>
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                   <td><p class="info-18">Construction  of the 3OC12HSL-dependent C4HSL production and chloramphenicol resistance  expression module</p>                  </td>
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                   <td>&nbsp;</td>
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                   <td><p class="info-18">We  created a symbiosis of Company E.coli and Customer E.coli for reproducing the situation  in real economy. We used signaling molecules and antibiotics resistance gene ,and  constructed signal-dependent signal production in our system.</p>                   </td>
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                   <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-4-1-1.png"><img src="https://static.igem.org/mediawiki/2014/a/a6/Tokyo_Tech_3-4-1-1.png" width="513" height="228" /></a></div></td>
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                   <td><p class="info-18">For  construction of the 3OC12HSL-dependent chloramphenicol resistance (CmR) and C4HSL  production module, we constructed a new part Plux-CmR-rhlI (BBa_K1529265). Plux-CmR-RhlI cell is an engineered E.coli  that contains a 3OC12HSL-dependent rhlI generator and a constitutive luxR  generator. We constructed a new Biobrick part Plux-CmR-rhlI by combining  Plux-CmR (BBa_K39562) and rhlI (BBa_). As a constitutive luxR generator, we used Pret-luxR (BBa_S0319). In our bank  story, this part is customer.</p>                   </td>
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                   <td><div align="center"><strong>Fig. 3-4-1-1. </strong>Genetic Circuit of Customer</div></td>
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                   <td><p class="head">1-1 3OC12HSL-dependent C4HSL production </p></td>
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                   <td>&nbsp;</td>
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                   <td><p class="info-18">First, we performed a reporter assay by using rhl reporter cell to characterize the function of  3OC12HSL-dependent C4HSL production. As the negative control of C4HSL  production, we prepared C4HSL non-producer cell. C4HSL non-producer cell  contains PlacIq-CmR instead of Plux-CmR-rhlI. The cell of negative control does  not produce C4HSL even in the presence of 3C12HSL.</p></td>
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                   <td>&nbsp;</td>
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                  <td class="entry-long"><a name="Summary" id="Summary"></a></td>
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                  <td>&nbsp;</td>
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                   <td><p class="info-18">Sender</p></td>
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                   <td><h2>2.  Summary of the experiments</h2></td>
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                   <td><h1>2-1. 3OC12HSL-dependent CmR expression</h1></td>
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                   <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-4-1-2.png"><img src="https://static.igem.org/mediawiki/2014/1/16/Tokyo_Tech_3-4-1-2.png" width="500" /></a></div></td>
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                   <td><div align="center"> <strong>Fig. 3-4-2-1.</strong> 3OC12HSL-dependent CmR expression assay flow chart                  </div></td>
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                  <td><p class="info-18">We confirmed the function of 3OC12HSL-dependent CmR expression by measuring the optical density of the cell cultures containing chloramphenicol.(Fig. 3-4-2-1) </p>
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                    <p class="info-18"> In this experiment, we prepared two plasmids A and B (See Fig. 3-4-2-2).Right after the 3OC12HSL induction, we added chloramphenicol into the medium including Customer cell. We measured the optical density for about eight hours to estimate the concentration of the cell. </p>
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                  <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-4-2-1.png"><img src="https://static.igem.org/mediawiki/2014/6/65/Tokyo_Tech_3-4-2-1.png" width="500" height="184" /></a></div></td>
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                  <td><div align="center"><strong>Fig. 3-4-2-2.</strong> Plasmids for the experiment of 3OC12HSL-dependent CmR expression</td>
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                   <td><p class="info-18">Repoter</p></td>
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                   <td><a name="2.2" id="2.2"></a></td>
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                  <td><h1>2-2. 3OC12HSL-dependent C4HSL production</h1></td>
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                  <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-4-1-3.png"><img src="https://static.igem.org/mediawiki/2014/b/b7/Tokyo_Tech_3-4-1-3.png" width="500" /></a></div></td>
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                  <td><div align="center"><strong>Fig. 3-4-2-3.</strong> 3OC12HSL-dependent C4HSL production assay flow chart</div></td>
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                   <td>&nbsp;</td>
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                   <td><p class="info-18">We performed a reporter assay by using reporter cell C, D and E to characterize the function of 3OC12HSL-dependent C4HSL production. Plux-CmR-RhlI cell containing constitutive LuxR generator expresses RhlI and produces C4HSL (RhlI) in the presence of 3OC12HSL. Since C4HSL is excreted to the culture, the supernatant of the sender cell contains C4HSL when this part works as expected.</td>
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                  <td><p class="info-18"> The reporter cell was incubated in the supernatant of the culture of the sender cell. When there are C4HSL in the supernatant, the reporter cell expresses GFP.(Fig. 3-4-2-3) We checked the fluorescence intensity of the reporter cell to confirm the production of C4HSL. The fluorescence intensity of the reporter cell was measured by flow cytometer.</p></td>
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                   <td class="info-18"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-4-2-2.png"><img src="https://static.igem.org/mediawiki/2014/8/8b/Tokyo_Tech_3-4-2-2.png" width="700"/></a></div></td>
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                   <td><div align="center"><strong>Fig. 3-4-2-4.</strong> Plasmids for the experiment of 3OC12HSL-dependent C4HSL production</div></td>
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                   <td><p class="info-18">We prepared four culture conditions as follow.</p></td>
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                   <td><p class="info-18">We prepared the following conditions for the induction of the reporter cells. (PlacIq-CmR cell was used as the negative control of RhlI. See Fig.3-4-2-4)</p></td>
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                   <td class="info-18">    A) Culture containing  Plux-CmR-RhlI cell with 3OC12HSL induction</td>
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                   <td class="info-18">&nbsp;</td>
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                   <td class="info-18">    B) Culture containing Plux-CmR-RhlI cell without 3OC12HSL induction</td>
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                   <td class="info-18">(1) Culture containing sender A (Plux-CmR-RhlI) with 3OC12HSL induction</td>
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                   <td class="info-18">    C) Culture containing Plux-CmR cell with 3OC12HSL induction</td>
+
                   <td class="info-18">(2) Culture containing sender A (Plux-CmR-RhlI) without induction
 +
                    </td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">    D) Culture containing Plux-CmR cell without 3OC12HSL induction</td>
+
                   <td class="info-18">(3) Culture containing sender B (PlacIq-CmR) with 3OC12HSL induction </td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><p class="info-18">The supernatants of this four different culture were  used as the inducer in the reporter assay. </p></td>
+
                   <td class="info-18">(4) Culture containing sender B (PlacIq-CmR) without induction </td>
 +
                </tr>
 +
                  <tr>
 +
                  <td class="info-18">(5)5 microM of synthetic C4HSL in LB medium  </td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
 +
                  <td class="info-18">(6) DMSO in LB medium </td>
 +
                </tr>
 +
                  <tr>
                   <td>&nbsp;</td>
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><p class="info-18">In the reporter assay, we used a Rhl reporter strain  that contains Ptet-rhlR and Plux-GFP. Also, a reporter cell that expresses GFP  constitutively and a reporter cell that does not express GFP were used as the  positive control and the negative control, respectively.</p></td>
+
                   <td><p class="info-18">Reporter cells</p></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td>&nbsp;</td>
+
                   <td class="info-18">&nbsp;</td>
 +
                </tr>               
 +
                <tr>
 +
                  <td class="info-18">C. The cell containing constitutive RhlR generator and Prhl(RL)-GFP</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><p class="head">1-2  3OC12HSL-dependent growth</p></td>
+
                   <td class="info-18">D. The cell containing constitutive RhlR generator and PlacUV5-GFP…Positive control</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><p class="info-18">The cell which contains Plux-CmR-rhlI can grow in the medium even containing chloramphenicol  .( Chloramphenicol is one of the antibiotics. )
+
                   <td class="info-18">E. The cell containing constitutive RhlR generator and Promoter-less-GFP…Negative control</td>
-
                  </p>                  </td>
+
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><p class="info-18">After  induction, we added chloramphenicol into the medium and measured optical  density hourly after induction. </p></td>
+
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
 +
                <tr>
 +
                  <td class="entry-long"><a name="Results" id="Results"></a></td>
 +
                </tr>   
                 <tr>
                 <tr>
                   <td>&nbsp;</td>
                   <td>&nbsp;</td>
 +
                </tr>                 
 +
                <tr>
 +
                  <td><h2>3.  Results</h2></td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;<a name="3.1" id="3.1"></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><h2>2. Results</h2></td>
+
                   <td><h1>3-1. 3OC12HSL-dependent CmR expression assay</h1></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
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                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><p class="head">2-1 3OC12HSL-dependent C4HSL production</p></td>
+
                   <td><p class="info-18">We tested two types of culture condition which contains different concentration of chloramphenicol(Cm). (0 and 100 microg / mL)</p>
 +
                  <p class="info-18">Fig. 3-4-3-1 and Fig. 3-4-3-2 show the condition in the absence and the presence of chloramphenicol, respectively.</p>
 +
                  <p class="info-18">Fig. 3-4-3-1 shows that every cell can grow in the absence of chloramphenicol.</p></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><p class="info-18">We measured GFP  expression in the reporter cell by flow cytometer</p></td>
+
                   <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-4-3-1.png"><img src="https://static.igem.org/mediawiki/2014/f/f1/Tokyo_Tech_3-4-3-1.png" width="700" /></a></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><p class="head">2-2 3OC12HSL-dependent growt</p>                   </td>
+
                   <td><div align="center"><strong>Fig. 3-4-3-1.</strong> 3OC12HSL-dependent customer growth in no chloramphenicol</div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><p class="info-18">After induction, optical density were measured to estimate the  concentration of the cell.</p></td>
+
                   <td><div align="center"></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-1-1.png"><img src="https://static.igem.org/mediawiki/2014/6/62/Tokyo_Tech_3-1-1.png" alt="" width="450" /></a></div></td>
+
                   <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-4-3-2.png"><img src="https://static.igem.org/mediawiki/2014/5/5d/Tokyo_Tech_3-4-3-2.png" width="700" /></a></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><div align="center">Fig. 3-2-1 </div></td>
+
                   <td><div align="center"><strong>Fig. 3-4-3-2.</strong> 3OC12HSL-dependent Customer growth in 100 microg / mL chloramphenicol</div></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><div align="center"></div></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="info-18">On the other hand, in the presence of chloramphenicol, the cell containing Plux-CmR-RhlI can grow only when it was induced by 3OC12HSL. Without the induction of 3OC12HSL, the cell cannot express CmR and cannot grow in the presence of chloramphenicol. As a result, we confirmed that Plux-CmR-RhlI expressed CmR when induced by 3OC12HSL as expected.</p></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
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                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-1-2.png"><img src="https://static.igem.org/mediawiki/2014/b/be/Tokyo_Tech_3-1-2.png" alt="" width="450" /></a></div></td>
+
                   <td><a name="3.2" id="3.2"></a>&nbsp;<a name="3.2" id="3.2"></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><div align="center">Fig. 3-2-2 </div></td>
+
                   <td><h1>3-2. 3OC12HSL-dependent C4HSL production assay</h1></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
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                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><p class="info-18">As Fig 3-2-shows, the cell containing Plux-CmR-RhlI can grow with induction of 3OC12SL, but can’t  without induction. Plux-CmR-RhlI cell grows more slowly than the positive  control. The amount of CmR is lower than the positive control, Compared Fig  3-2-1 to Fig 3-2-2, With Cm, the cell grow more slowly than without Cm.  geExpression of CmR in Plux-CmR-RhlI depende  on induction by adding 2OC12HSL.          </p>                   </td>
+
                   <td><p class="info-18">Fig. 3-4-3-3 shows the fluorescence intensities generated by reporter cells. When the reporter cell C (Plux-CmR-RhlI) was incubated in the condition (1) (the culture of the induced Customer cell), the fluorescence intensity of the reporter cell increased. Comparing the results of condition (1) and (2), reporter cell in the supernatant of (1) had 95-fold higher fluorescence intensity.</p>
 +
                  <p class="info-18">This result indicates that Customer cell produced C4HSL in response to 3OC12HSL induction by the function of Plux-CmR-RhlI.</p>
 +
                  <p class="info-18">From this experiment, we confirmed that a new part Plux-CmR-RhlI synthesized C4HSL (RhlI) as expected.</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-4-3-3.png"><img src="https://static.igem.org/mediawiki/2014/d/d6/Tokyo_Tech_3-4-3-3.png" width="500" height="320" /></a></div></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><div align="center"><strong>Fig. 3-4-3-3.</strong> Customer excretes C4HSL when C12HSL exists</div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
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                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><h2>3.  Materials and methods</h2></td>
+
                   <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="entry-long">&nbsp;<a name="Materials" id="Materials"></a></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
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                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><p class="head">3-1 Construction</p></td>
+
                   <td><h2>4. Materials and methods</h2></td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;<a name="4.1" id="4.1"></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><h1>4-1 Construction</h1></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
Line 223: Line 326:
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><p class="info-18">All  the samples were JM2.300 strain</p></td>
+
                   <td><p class="info-18">All  the samples were JM2.300 strain.</p>                   </td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
                   <td><p class="head">-Plasmids</p></td>
                   <td><p class="head">-Plasmids</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td style="text-indent:0px;"><h3>3OC12HSL-dependent CmR expression</h3></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
Line 232: Line 338:
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><p class="head">3-2 Protocol </p></td>
+
                   <td class="info-18"><div align="left">A.Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3) </div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><p class="info-18" style="text-indent:0px;">3-2-1.  3OC12HSL-dependent C4HSL production assay by using reporter assay</p></td>
+
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><p class="info-18"><strong>Prepare the supernatant of the sender cell</strong></p></td>
+
                   <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-4-4-1.png"><img src="https://static.igem.org/mediawiki/2014/5/5f/Tokyo_Tech_3-4-4-1.png" width="500" /></a></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">1. Grow the colony of sender cell in LB containing antibiotic  O/N at 37°C.</td>
+
                   <td><div align="center"> <strong>Fig. 3-4-4-1. </strong></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">2. Make a 1:100 dilution in 3 mL of fresh LB containing  antibiotic and grow the cells at 37°C <br />
+
                   <td><div align="center"></div></td>
-
          until the observed OD590 reaches 0.5.</td>
+
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">3. Centrifuge  1mL of the sample at 5000g, RT for 1 minute.</td>
+
                   <td class="info-18">B. Ptet-GFP-Ptet-RhlR (pSB6A1), PlacIq-CmR (pSB3K3)...Positive control</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">4. Suspend the pellet in <u>1 mL of LB containing Ampicillin(50μg/mL)and Kanamycin (30μg/mL)</u></td>
+
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">5. Add 30&#181;L of suspension in the following medium.</td>
+
                   <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-4-4-2.png"><img src="https://static.igem.org/mediawiki/2014/7/74/Tokyo_Tech_3-4-4-2.png" width="500" /></a></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">           Add 3&#181;L of 5&#181;M C12HSL to 3mL LB containing Amp and Kan</td>
+
                   <td><div align="center"><strong>Fig.  3-4-4-2.</strong></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">           Add 3&#181;L DMSO to 3 &#181;M of LB containing Amp and Kan</td>
+
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
-
               
 
                 <tr>
                 <tr>
-
                   <td class="info-18">6. Grow  the samples of sender cell at 37°C for 4 hours.</td>
+
                   <td><h3>3OC12HSL-dependent C4HSL production</h3></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">7. Measure optical density every hour (If optical  density is over 1.0, dilute the cell medium.)</td>
+
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">8. Centrifuge  sample at 9000g, 4°C for 1minute.Filter sterilize supernatant.</td>
+
                   <td class="info-18" style="text-indent:0px;"><strong>Sender:</strong></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">9. Use  the supernatant in reporter assay</td>
+
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">&nbsp;</td>
+
                   <td class="info-18">A. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td><p class="info-18"><strong>Reporter Assay</strong></p></td>
+
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">1. Grow the colony of Reporter cell(D~F)  in LB containing antibiotic O/N at 37°C.</td>
+
                   <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-4-4-3.png"><img src="https://static.igem.org/mediawiki/2014/8/88/Tokyo_Tech_3-4-4-3.png" width="500" /></a></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">2. Make a 1:100 dilution in 3 mL of fresh LB+ antibiotic and grow the cells at 37C until you reach an 0.5 OD590.(fresh  culture)</td>
+
                   <td><div align="center"><strong>Fig3-4-4-3.</strong></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">3. Centrifuge  sample at 5000g, 25°C RT for 1 minute. Discard the supernatant.</td>
+
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">4. Suspended  the sample in 3 mL of LB containing Ampicillin(50μg/mL) and Kanamycin(30μg/mL)</td>
+
                   <td class="info-18">B. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR(pSB3K3)...Negative control</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">5. Add 30&#181;L of suspension in the following medium.</td>
+
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">           Filtrate  of A①+3mL of  LB containing Amp and Kan</td>
+
                   <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-4-4-4.png"><img src="https://static.igem.org/mediawiki/2014/8/8b/Tokyo_Tech_3-4-4-4.png" width="500" /></a></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">           Filtrate of A②+3mL of  LB containing Amp and Kan</td>
+
                   <td><div align="center"><strong>Fig. 3-4-4-4.</strong></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">           Filtrate  of B①+3mL of  LB containing Amp and Kan</td>
+
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">           Filtrate  of B②+3mL of  LB containing Amp and Kan</td>
+
                   <td class="info-18" style="text-indent:0px;"><strong>Reporter:</strong></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">           Filtrate  of C①+3mL of  LB containing Amp and Kan</td>
+
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">          Filtrate  of C②+3mL of  LB containing Amp and Kan</td>
+
                   <td class="info-18"><p align="left" class="info-18">C. Ptet-RhlR(pSB6A1), Plux-GFP(pSB3K3)</p></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">          C4HSL+3mL of LB containing Amp and Kan</td>
+
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">          DMSO +  3mL of LB containing Amp and Kan</td>
+
                   <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-4-4-5.png"><img src="https://static.igem.org/mediawiki/2014/b/b7/Tokyo_Tech_3-4-4-5.png" width="500" /></a></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">6. Grow the samples of Reporter cell in incubator at 37°C for 4 hours.</td>
+
                   <td><div align="center"><strong>Fig3-4-4-5.</strong></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">7. Start  preparing the flow cytometer 1 h before the end of incubation.</td>
+
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">8. Take  200 microL of the sample, and centrifuge at 9000 Xg, 1 min, 4°C.</td>
+
                   <td class="info-18">D. Ptet-RhlR(pSB6A1), PlacUV5-GFP(pSB3K3)...Positive control</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">9. Remove  the supernatant by using P1000 pipette.</td>
+
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">10. Add 1 mL  of filtered PBS (phosphate-buffered saline) and suspend.</td>
+
                   <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-4-4-6.png"><img src="https://static.igem.org/mediawiki/2014/a/a8/Tokyo_Tech_3-4-4-6.png" width="500"  /></a></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">11. Dispense  all of each suspension into a disposable tube through a cell strainer.</td>
+
                   <td><div align="center"><strong>Fig. 3-4-4-6.</strong></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">12. Use  flow cytometer to measure the fluorescence  of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton,  Dickenson and Company).</td>
+
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">&nbsp;</td>
+
                   <td class="info-18">E. Ptet-RhlR(pSB6A1), Promoter-less-GFP(pSB3K3)...Negative control</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="head"><p>3-2-2. 3OC12HSL-depemdent CmR expression</p></td>
+
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">1. Grow the colony of sender cell in LB containing  antibiotic O/N at 37°C.</td>
+
                   <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-4-4-7.png"><img src="https://static.igem.org/mediawiki/2014/b/b3/Tokyo_Tech_3-4-4-7.png" width="500" /></a></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">2. Make a 1:100 dilution in 3 mL of fresh LB containig  antibiotic and grow the cells at 37°C until the observed OD590 reaches  0.5.(fresh culture)</td>
+
                   <td><div align="center"><strong>Fig. 3-4-4-7.</strong></div></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">3. Centrifuge  1mL of the sample at 5000g, RT for 1 minute.</td>
+
                   <td>&nbsp;</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">4. Suspend the pellet in <u>1 mL of LB containing  Ampicillin(50 microg/mL)and Kanamycin(30 microg/mL)</u></td>
+
                   <td><a name="4.2" id="4.2"></a></td>
 +
                </tr>               
 +
                <tr>
 +
                  <td><h1>4-2. Assay Protocol </h1><a name="4.2.1" id="4.2.1"></td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">5. Add 30&#181;L of suspension in the following medium.</td>
+
                   <td><p class="info-18" style="text-indent:0px;"><strong>4-2-1. 3OC12HSL-depemdent CmR expression</strong></p></td>
                 </tr>
                 </tr>
 +
               
                 <tr>
                 <tr>
-
                   <td class="info-18">           Add 3&#181;L of 5&#181;M C12HSL to 3mL LB containing Amp,Kan(concentration  is described upper) and Cm(100 microg /mL)</td>
+
                   <td class="info-18">1.Prepare the overnight culture of cell A and B at 37°C.</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">           Add 3&#181; DMSO to 3mL  of LB containing Amp and Kan</td>
+
                   <td class="info-18">2.Make a 1:100 dilution in 3 mL of fresh LB containing antibiotics and grow the cell at 37°C until the observed OD590 reaches 0.5 (→fresh culture) </td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">6. Grow  the samples of sender cell at 37°C for 4 hours.</td>
+
                   <td class="info-18">3. Add 30 microL of suspension in the following medium.</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">7. Measure optical density every hour.  (If optical density is over 1.0, dilute the  cell medium.)</td>
+
                   <td colspan="2" class="info-18" style="text-indent:50px;">1) 3 mL of LB containing Amp and Kan + 30 microL C4HSL (final concentration is 500 microM)</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
-
                   <td class="info-18">&nbsp;</td>
+
                   <td colspan="2" class="info-18" style="text-indent:50px;">2) 3 mL of LB containing Amp and Kan + 30 microL DMSO</td>
                 </tr>
                 </tr>
 +
               
                 <tr>
                 <tr>
-
                   <td><h2>4. Reference</h2></td>
+
                   <td colspan="2" class="info-18" style="text-indent:50px;">3) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100microg / mL) + 30 microL C4HSL  (final concentration is 500 microM)</td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2" class="info-18" style="text-indent:50px;">4) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100microg / mL) + 30 microL DMSO</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">4. Grow the samples of sender cells at 37°C for more than 10 hours. Measure optical density every hour. (If optical density is over 1.0, dilute the cell medium.)</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;<a name="4.2.2" id="4.2.2"></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="info-18" style="text-indent:0px;"><strong>4-2-2. 3OC12HSL-dependent C4HSL production</strong></p></td>
 +
                </tr>
 +
               
 +
                <tr>
 +
                  <td class="info-18">Prepare the supernatant of the sender cell</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">1. Grow the colony of sender cell in LB containing antibiotic O/N at 37°C.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">2. Make a 1:100 dilution in 3 mL of fresh LB containing antibiotic and grow the cells at 37°C until the observed OD590 reaches 0.5.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">3. Add 30 microL of the culture containing the cells in the following medium.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2" class="info-18" style="text-indent:50px;">a) Add 30 microL of 500 microM 3OC12HSL to 3 mL LB containing Amp and Kan</td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2" class="info-18" style="text-indent:50px;">b) Add 30 microL DMSO to 3 mL LB containing Amp and Kan</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">4 .Grow the samples of sender cell at 37°C for 8 hours.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">5. Centrifuge sample at 9000x g, 4°C for 1minute. Filter sterilize supernatant. (Pore size is 0.22 microm. ) Use this supernatant in reporter assay.</td>
                 </tr>
                 </tr>
                 <tr>
                 <tr>
                   <td class="info-18">&nbsp;</td>
                   <td class="info-18">&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18" style="text-indent:0px;"><strong>Reporter Assay</strong></td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">1. Grow the colony of Reporter cell (described upper) in LB containing antibiotic (Amp and Kan) over night at 37°C.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">2. Make a 1:100 dilution in 3 mL of fresh LB+ antibiotics and grow the cells at 37°C until you reach an 0.5 in OD590 (fresh culture).</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">3. Add 30 microL of the culture containing reporter cell in the following medium.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2" class="info-18" style="text-indent:50px;">1) 2.7 mL filtrate of Aa +300 microL LB</td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2" class="info-18" style="text-indent:50px;">2) 2.7 mL filtrate of Ab +300 microL LB</td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2" class="info-18" style="text-indent:50px;">3) 2.7 mL filtrate of Ba +300 microL LB</td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2" class="info-18" style="text-indent:50px;">4) 2.7 mL filtrate of Bb +300 microL LB</td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2" class="info-18" style="text-indent:50px;">5) 3 mL LB + 500 microM C4HSL 30 microM (final concentration is 5 microM)</td>
 +
                </tr>
 +
                <tr>
 +
                  <td colspan="2" class="info-18" style="text-indent:50px;">6) 3 mL LB + DMSO 30 microL</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">4. Grow the samples of Reporter cell in incubator at 37°C for 4 hours.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">5. Start preparing the flow cytometer 1 h before the end of incubation.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">6. After incubation, take the sample, and centrifuge at 9000x g, 1 min, 4°C.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">7. Remove the supernatant by using P1000 pipette.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">8. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3.)</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">9. Dispense all of each suspension into a disposable tube through a cell strainer.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">10. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="entry-long"><a name="Reference" id="Reference"></a></td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td><h2>5. Reference</h2></td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">1. Bo Hu <em>et al.</em> (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619</td>
                 </tr>
                 </tr>
               </table>
               </table>
               <p>&nbsp;</p>
               <p>&nbsp;</p>
-
            </div>
+
          </div>
-
            </div>
+
        </div>
  </div>
  </div>
<!-- end #content -->
<!-- end #content -->

Latest revision as of 03:41, 18 October 2014

Tokyo_Tech

Experiment

3OC12HSL-dependent C4HSL production

 

Contents

1. Introduction

2. Summary of the Experiments

2-1. 3OC12HSL-dependent CmR expression

2-2. 3OC12HSL-dependent C4HSL production

3. Results

3.1. 3OC12HSL-dependent CmR expression

3.2. 3OC12HSL-dependent C4HSL production

4. Materials and Methods

4.1. Construction

4.2. Assay Protocol

4.2.1. 3OC12HSL-depemdent CmR expression

4.2.2. C4HSL-Dependent 3OC12HSL Production Assay

5. Reference

 
 

1. Introduction

 

We designed a signal-dependent signal production in our system by using signaling molecules and antibiotics resistance gene. In our bank story, we used signaling molecule 3OC12HSL as product.

For construction of the 3OC12HSL-dependent chloramphenicol resistance gene product(CmR) and C4HSL production module, we constructed a new part Plux-CmR-RhlI (BBa_K1529797). Plux-CmR-RhlI cell is an engineered E. coli that contains 3OC12HSL-dependent RhlI generator and a constitutive LuxR generator. As a constitutive LuxR generator, we used Ptet-LuxR. In our bank story, this part imitates the function of Customer. (Fig. 3-4-1-1) We confirmed the 3OC12HSL-dependent growth by measuring the optical density, and 3OC12HSL-dependent C4HSL production by using reporter cell.

 
Fig. 3-4-1-1. Genetic Circuit of Customer
 
 
 
 

2. Summary of the experiments

 

2-1. 3OC12HSL-dependent CmR expression

Fig. 3-4-2-1. 3OC12HSL-dependent CmR expression assay flow chart

We confirmed the function of 3OC12HSL-dependent CmR expression by measuring the optical density of the cell cultures containing chloramphenicol.(Fig. 3-4-2-1)

In this experiment, we prepared two plasmids A and B (See Fig. 3-4-2-2).Right after the 3OC12HSL induction, we added chloramphenicol into the medium including Customer cell. We measured the optical density for about eight hours to estimate the concentration of the cell.

Fig. 3-4-2-2. Plasmids for the experiment of 3OC12HSL-dependent CmR expression
 

2-2. 3OC12HSL-dependent C4HSL production

Fig. 3-4-2-3. 3OC12HSL-dependent C4HSL production assay flow chart
 

We performed a reporter assay by using reporter cell C, D and E to characterize the function of 3OC12HSL-dependent C4HSL production. Plux-CmR-RhlI cell containing constitutive LuxR generator expresses RhlI and produces C4HSL (RhlI) in the presence of 3OC12HSL. Since C4HSL is excreted to the culture, the supernatant of the sender cell contains C4HSL when this part works as expected.

The reporter cell was incubated in the supernatant of the culture of the sender cell. When there are C4HSL in the supernatant, the reporter cell expresses GFP.(Fig. 3-4-2-3) We checked the fluorescence intensity of the reporter cell to confirm the production of C4HSL. The fluorescence intensity of the reporter cell was measured by flow cytometer.

Fig. 3-4-2-4. Plasmids for the experiment of 3OC12HSL-dependent C4HSL production
 

We prepared the following conditions for the induction of the reporter cells. (PlacIq-CmR cell was used as the negative control of RhlI. See Fig.3-4-2-4)

 
(1) Culture containing sender A (Plux-CmR-RhlI) with 3OC12HSL induction
(2) Culture containing sender A (Plux-CmR-RhlI) without induction
(3) Culture containing sender B (PlacIq-CmR) with 3OC12HSL induction
(4) Culture containing sender B (PlacIq-CmR) without induction
(5)5 microM of synthetic C4HSL in LB medium
(6) DMSO in LB medium
 

Reporter cells

 
C. The cell containing constitutive RhlR generator and Prhl(RL)-GFP
D. The cell containing constitutive RhlR generator and PlacUV5-GFP…Positive control
E. The cell containing constitutive RhlR generator and Promoter-less-GFP…Negative control
 
 

3. Results

 

3-1. 3OC12HSL-dependent CmR expression assay

 

We tested two types of culture condition which contains different concentration of chloramphenicol(Cm). (0 and 100 microg / mL)

Fig. 3-4-3-1 and Fig. 3-4-3-2 show the condition in the absence and the presence of chloramphenicol, respectively.

Fig. 3-4-3-1 shows that every cell can grow in the absence of chloramphenicol.

Fig. 3-4-3-1. 3OC12HSL-dependent customer growth in no chloramphenicol
Fig. 3-4-3-2. 3OC12HSL-dependent Customer growth in 100 microg / mL chloramphenicol

On the other hand, in the presence of chloramphenicol, the cell containing Plux-CmR-RhlI can grow only when it was induced by 3OC12HSL. Without the induction of 3OC12HSL, the cell cannot express CmR and cannot grow in the presence of chloramphenicol. As a result, we confirmed that Plux-CmR-RhlI expressed CmR when induced by 3OC12HSL as expected.

 
 

3-2. 3OC12HSL-dependent C4HSL production assay

 

Fig. 3-4-3-3 shows the fluorescence intensities generated by reporter cells. When the reporter cell C (Plux-CmR-RhlI) was incubated in the condition (1) (the culture of the induced Customer cell), the fluorescence intensity of the reporter cell increased. Comparing the results of condition (1) and (2), reporter cell in the supernatant of (1) had 95-fold higher fluorescence intensity.

This result indicates that Customer cell produced C4HSL in response to 3OC12HSL induction by the function of Plux-CmR-RhlI.

From this experiment, we confirmed that a new part Plux-CmR-RhlI synthesized C4HSL (RhlI) as expected.

Fig. 3-4-3-3. Customer excretes C4HSL when C12HSL exists
 
 
 
 

4. Materials and methods

 

4-1 Construction

-Strain

All the samples were JM2.300 strain.

-Plasmids

3OC12HSL-dependent CmR expression

 
A.Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)
 
Fig. 3-4-4-1.
B. Ptet-GFP-Ptet-RhlR (pSB6A1), PlacIq-CmR (pSB3K3)...Positive control
 
Fig. 3-4-4-2.
 

3OC12HSL-dependent C4HSL production

 
Sender:
 
A. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)
 
Fig. 3-4-4-3.
 
B. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR(pSB3K3)...Negative control
 
Fig. 3-4-4-4.
 
Reporter:
 

C. Ptet-RhlR(pSB6A1), Plux-GFP(pSB3K3)

 
Fig. 3-4-4-5.
 
D. Ptet-RhlR(pSB6A1), PlacUV5-GFP(pSB3K3)...Positive control
 
Fig. 3-4-4-6.
 
E. Ptet-RhlR(pSB6A1), Promoter-less-GFP(pSB3K3)...Negative control
 
Fig. 3-4-4-7.
 

4-2. Assay Protocol

4-2-1. 3OC12HSL-depemdent CmR expression

1.Prepare the overnight culture of cell A and B at 37°C.
2.Make a 1:100 dilution in 3 mL of fresh LB containing antibiotics and grow the cell at 37°C until the observed OD590 reaches 0.5 (→fresh culture)
3. Add 30 microL of suspension in the following medium.
1) 3 mL of LB containing Amp and Kan + 30 microL C4HSL (final concentration is 500 microM)
2) 3 mL of LB containing Amp and Kan + 30 microL DMSO
3) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100microg / mL) + 30 microL C4HSL (final concentration is 500 microM)
4) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100microg / mL) + 30 microL DMSO
4. Grow the samples of sender cells at 37°C for more than 10 hours. Measure optical density every hour. (If optical density is over 1.0, dilute the cell medium.)
 

4-2-2. 3OC12HSL-dependent C4HSL production

Prepare the supernatant of the sender cell
1. Grow the colony of sender cell in LB containing antibiotic O/N at 37°C.
2. Make a 1:100 dilution in 3 mL of fresh LB containing antibiotic and grow the cells at 37°C until the observed OD590 reaches 0.5.
3. Add 30 microL of the culture containing the cells in the following medium.
a) Add 30 microL of 500 microM 3OC12HSL to 3 mL LB containing Amp and Kan
b) Add 30 microL DMSO to 3 mL LB containing Amp and Kan
4 .Grow the samples of sender cell at 37°C for 8 hours.
5. Centrifuge sample at 9000x g, 4°C for 1minute. Filter sterilize supernatant. (Pore size is 0.22 microm. ) Use this supernatant in reporter assay.
 
Reporter Assay
1. Grow the colony of Reporter cell (described upper) in LB containing antibiotic (Amp and Kan) over night at 37°C.
2. Make a 1:100 dilution in 3 mL of fresh LB+ antibiotics and grow the cells at 37°C until you reach an 0.5 in OD590 (fresh culture).
3. Add 30 microL of the culture containing reporter cell in the following medium.
1) 2.7 mL filtrate of Aa +300 microL LB
2) 2.7 mL filtrate of Ab +300 microL LB
3) 2.7 mL filtrate of Ba +300 microL LB
4) 2.7 mL filtrate of Bb +300 microL LB
5) 3 mL LB + 500 microM C4HSL 30 microM (final concentration is 5 microM)
6) 3 mL LB + DMSO 30 microL
4. Grow the samples of Reporter cell in incubator at 37°C for 4 hours.
5. Start preparing the flow cytometer 1 h before the end of incubation.
6. After incubation, take the sample, and centrifuge at 9000x g, 1 min, 4°C.
7. Remove the supernatant by using P1000 pipette.
8. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3.)
9. Dispense all of each suspension into a disposable tube through a cell strainer.
10. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)
 
 
 

5. Reference

1. Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619