Team:Tokyo Tech/Experiment/C4HSL-dependent 3oxoC12HSL production

From 2014.igem.org

(Difference between revisions)
Line 14: Line 14:
<!-- end #header -->
<!-- end #header -->
-
        <div id="menu">
+
<div id="page">
-
<ul>
+
-
<li><a href="https://2014.igem.org/Team:Tokyo_Tech">Home</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Project">Project</a></li>
+
-
<li class="current_page_item"><a href="#">Experiment</a>
+
-
  <ul>
+
-
        <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>
+
-
        <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>
+
-
        <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>
+
-
        <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>
+
-
+
-
  </ul>
+
-
</li>
+
-
<li><a href="#">Modeling</a>
+
-
                            <ul>
+
-
                                <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Modeling/Modeling1" style="width:260px; margin-left:-63px;">なぞなぞ</a></li>
+
-
                                <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Modeling/Modeling2" style="width:260px; margin-left:-63px;">Parameter sensitivity analysis</a></li>
+
-
                                <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>
+
-
                            </ul>
+
-
                        </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:45px; padding-top:4px;">Policy and Practices</a></li>
+
-
<li><a href="#">More</a>
+
-
    <ul>
+
-
<li><a href="https://2014.igem.org/Team:Tokyo_Tech/About_Us" style="width:240px; margin-left:-53px;">About Us</a></li>
+
-
        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Safety" style="width:240px; margin-left:-53px;">Safety</a></li>
+
-
        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Attribution_and_Contribution" style="width:240px; margin-left:-53px;">Attribution and Contribution</a></li>
+
-
        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Collaboration" style="width:240px; margin-left:-53px;">Collaboration</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://igem.org/Team.cgi?id=1529" style="width:240px; margin-left:-53px;">Official Team Page</a></li>
+
-
    </ul>
+
-
</li>
+
-
</ul>
+
-
      </div>
+
-
<!-- end #menu -->
+
-
+
-
<div id="content-header">
+
-
+
-
        </div>
+
-
+
-
<!-- end #content-header -->
+
-
+
-
 
+
-
<div id="page">
+
  <div id="content-over">
  <div id="content-over">
             <div class="post">
             <div class="post">
<h2 class="title">Experiment</h2>
<h2 class="title">Experiment</h2>
-
                 <span class="meta">C4HSL-dependent 3oxoC12HSL production</span>
+
                 <span class="meta">C4HSL-dependent 3OC12HSL production</span>
-
    <div class="entry-long">
+
                <div class="entry-long">
-
<p>Under Construction！</p>
+
              <p>&nbsp;</p>
-
<p>&nbsp;</p>
+
              <table width="900" border="0">
-
<p>&nbsp;</p>
+
                <tr>
-
<p>&nbsp;</p>
+
                  <td width="890"><div align="center" class="title-small">C4HSL-dependent 3OC12HSL production module</div></td>
-
<p>&nbsp;</p>
+
                </tr>
-
<p>&nbsp;</p>
+
                <tr>
-
<div align="center"><img src="http://www.actmp2014.com/images/under_construction%20(1).png" /></div>
+
                  <td>&nbsp;</td>
-
<p>&nbsp;</p>
+
                </tr>
-
<p>&nbsp;</p>
+
                <tr>
-
<p>&nbsp;</p>
+
                  <td>&nbsp;</td>
-
<p>&nbsp;</p>
+
                </tr>
-
<p>&nbsp;</p>
+
                <tr>
-
<p>&nbsp;</p>
+
                  <td><h2>1. Summary of the experiment </h2></td>
-
       
+
                </tr>
-
  </div>
+
                <tr>
-
    </div>
+
                  <td><p class="info-18">Construction of the C4HSL-dependent 3OC12HSL production and chloramphenicol resistance expression module.</p>                  </td>
-
+
                </tr>
-
  </div>
+
                <tr>
 +
                  <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>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="info-18">For construction of the C4HSL-dependent chloramphenicol resistance (CmR) and 3OC12HSL production module, we constructed a new part Plux-CmR-lasI (BBa_). Plux-CmR-lasI cell is an engineered E.coli that contains a C4HSL-dependent lasI generator and a constitutive rhlR generator. We constructed a new Biobrick part Plux-CmR-lasI by combining Plux-CmR (BBa_K39562) and lasI (BBa_). As a constitutive rhlR generator, we used Pret-rhlR (BBa_S0319). In our bank story, this part is company.                  </p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="head">1-1 C4HSL-dependent 3OC12HSL production </p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="info-18">First, we performed a reporter assay by using Lux reporter cell to characterize the function of C4HSL-dependent 3OC12HSL production. As the negative control of 3OC12HSL production, we prepared 3OC12HSL non-producer cell. 3OC12HSL non-producer cell contains Plux-CmR instead of Plux-CmR-lasI. The cell of negative control does not produce 3OC12HSL even in the presence of C4HSL.</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="info-18">Sender</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td><div align="center"><img src="http://sg.openrice.com/images/v4/previewimg/sr1-icon-noResult.png" alt="" width="600" /></div></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><div align="center"><img src="http://sg.openrice.com/images/v4/previewimg/sr1-icon-noResult.png" alt="" width="600" /></div></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><div align="center"><img src="http://sg.openrice.com/images/v4/previewimg/sr1-icon-noResult.png" alt="" width="600" /></div></td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="info-18">Repoter</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td><div align="center"><img src="http://sg.openrice.com/images/v4/previewimg/sr1-icon-noResult.png" alt="" width="600" /></div></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><div align="center"><img src="http://sg.openrice.com/images/v4/previewimg/sr1-icon-noResult.png" alt="" width="600" /></div></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><div align="center"><img src="http://sg.openrice.com/images/v4/previewimg/sr1-icon-noResult.png" alt="" width="600" /></div></td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="info-18">We prepared four culture conditions as follow.</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">    A) Culture containing Plux-CmR-LasI cell with C4HSL induction</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">    B) Culture containing Plux-CmR-LasI cell without C4HSL induction</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">    C) Culture containing Plux-CmR cell with C4HSL induction</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">    D) Culture containing Plux-CmR cell without C4HSL induction</td>
 +
                </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>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="info-18">In the reporter assay, we used a Lux reporter strain that contains Ptet-luxR 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>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="head">1-2  C4HSL-dependent growth</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="info-18">The cell which contains Plux-CmR-lasI can grow in the medium containing chloramphenicol<br />
 +
                  (Chloramphenicol is one of the antibiotics. )                  </p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="info-18">After induction, we added chloramphenicol into the medium and measured optical density<br />
 +
                  after induction.</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td><h2>2.  Results</h2></td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="head">2-1  C4HSL-dependent 3OC12HSL production</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="info-18">We measured the expression of GFP in the reporter cell by flow cytometer.</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="head">2-2 C4HSL-dependent growth</p>                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="info-18">After induction, optical density were measured to estimate the concentration of the cell.</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-1-1.png"><img src="http://sg.openrice.com/images/v4/previewimg/sr1-icon-noResult.png" alt="" width="600" /></a></div></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><div align="center">Fig. 3-1-1 </div></td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-1-2.png"><img src="http://sg.openrice.com/images/v4/previewimg/sr1-icon-noResult.png" alt="" width="600" /></a></div></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><div align="center">Fig. 3-1-2 </div></td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="info-18">explanation?    </p>                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td><h2>3.  Materials and methods</h2></td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="head">3-1  Construction</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="head">-Strain</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="info-18">All the samples were JM2.300 strain</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="head">-Plasmids</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td>&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="head">3-2 </p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="info-18" style="text-indent:0px;">3-2-1.  C4HSL-dependent 3OC12HSL production assay by using reporter assay</p></td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="info-18">Prepare the supernatant of the sender cell</p></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 37C until the observed OD590 reaches 0.5. </td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">3. Centrifuge 1mL of the sample at 5000g, RT for 1 minute.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">4. Suspend the pellet in<u> </u><u>1 mL of LB containing Ampicillin</u><u>（</u><u>50μg/mL</u><u>）</u><u>and Kanamycin(30μg/mL) .</u></td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">5. Add 30&#181;L of suspension in the following medium.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">           Add 30&#181;L of 500&#181;M C12HSL to 3mL LB containing Amp and Kan</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">           Add 30&#181;L DMSO to 3mL of LB containing Amp and Kan                  </td>
 +
                </tr>
 +
               
 +
                <tr>
 +
                  <td class="info-18">6. Grow the samples of sender cell at 37°C for 4 hours.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">7. Measure optical density every hour.  (If optical density is over 1.0, dilute the cell medium.)</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">8. Centrifuge sample at 9000g, 4°C for 1minute. Filter sterilize supernatant.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">9. Use  the supernatant in reporter assay.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td><p class="info-18"><strong>Reporter Assay</strong></p></td>
 +
                </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>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">2. Make a 1:100 dilution in 3 mL of fresh LB+  antibiotic and grow the cells at 37°C until you reach an 0.5 OD590. (fresh  culture).</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">3. Centrifuge  sample at 5000g, 25°C RT for 1 minute. Discard the supernatant.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">4. Suspended the sample in 3 mL of LB containing Ampicillin(50μg/mL) and Kanamycin(30μg/mL).</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">5. Add 30&#181;L of suspension in the following medium.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">           Filtrate  of A①＋3mL of  LB containing Amp and Kan</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">           Filtrate  of A②＋3mL of  LB containing Amp and Kan</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">           Filtrate  of B①＋3mL of  LB containing Amp and Kan</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">           Filtrate  of B②＋3mL of  LB containing Amp and Kan</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">           Filtrate  of C①＋3mL of  LB containing Amp and Kan</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">          Filtrate  of C②＋3mL of  LB containing Amp and Kan</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">          3OC12HSL＋3mL of LB containing Amp and Kan</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">          DMSO +  3mL of LB containing Amp and Kan</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">6. Grow  the samples of Reporter cell in incubator at 37°C for 4 hours.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">7. Start  preparing the flow cytometer 1 h before the end of incubation.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">8. Take  200 microL of the sample, and centrifuge at 9000 Xg, 1 min, 4°C.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">9. Remove  the supernatant by using P1000 pipette.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">10. Add 1 mL  of filtered PBS (phosphate-buffered saline) and suspend.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">11. Dispense  all of each suspension into a disposable tube through a cell strainer.</td>
 +
                </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>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="head"><p>3-2-2. C4HSL-depemdent CmR expression</p></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 containig  antibiotic and grow the cells at 37°C until the observed OD590 reaches  0.5 (fresh culture).</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">3. Centrifuge  1mL of the sample at 5000g, RT for 1 minute.</td>
 +
                </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>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">5. Add 30&#181;L of suspension in the following medium.</td>
 +
                </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>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">           Add 3microL DMSO to 3mL  of LB containing Amp and Kan.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">6. Grow  the samples of sender cell at 37°C for 4 hours.</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">7. Measure optical density every hour.  (If optical density is over 1.0, dilute the  cell medium.)</td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">&nbsp;</td>
 +
                </tr>
 +
                <tr>
 +
                  <td><h2>4. Reference</h2></td>
 +
                </tr>
 +
                <tr>
 +
                  <td class="info-18">&nbsp;</td>
 +
                </tr>
 +
              </table>
 +
              <p>&nbsp;</p>
 +
            </div>
 +
            </div>
 +
  </div>
<!-- end #content -->
<!-- end #content -->

Revision as of 14:09, 10 October 2014

Tokyo_Tech

Experiment

C4HSL-dependent 3OC12HSL production

 

C4HSL-dependent 3OC12HSL production module
 
 

1. Summary of the experiment

Construction of the C4HSL-dependent 3OC12HSL production and chloramphenicol resistance expression module.

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.

For construction of the C4HSL-dependent chloramphenicol resistance (CmR) and 3OC12HSL production module, we constructed a new part Plux-CmR-lasI (BBa_). Plux-CmR-lasI cell is an engineered E.coli that contains a C4HSL-dependent lasI generator and a constitutive rhlR generator. We constructed a new Biobrick part Plux-CmR-lasI by combining Plux-CmR (BBa_K39562) and lasI (BBa_). As a constitutive rhlR generator, we used Pret-rhlR (BBa_S0319). In our bank story, this part is company.

1-1 C4HSL-dependent 3OC12HSL production

First, we performed a reporter assay by using Lux reporter cell to characterize the function of C4HSL-dependent 3OC12HSL production. As the negative control of 3OC12HSL production, we prepared 3OC12HSL non-producer cell. 3OC12HSL non-producer cell contains Plux-CmR instead of Plux-CmR-lasI. The cell of negative control does not produce 3OC12HSL even in the presence of C4HSL.
 

Sender
 
 

Repoter
 
 

We prepared four culture conditions as follow.
    A) Culture containing Plux-CmR-LasI cell with C4HSL induction
    B) Culture containing Plux-CmR-LasI cell without C4HSL induction
    C) Culture containing Plux-CmR cell with C4HSL induction
    D) Culture containing Plux-CmR cell without C4HSL induction

The supernatants of this four different culture were used as the inducer in the reporter assay.
 

In the reporter assay, we used a Lux reporter strain that contains Ptet-luxR 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.
 

1-2 C4HSL-dependent growth

The cell which contains Plux-CmR-lasI can grow in the medium containing chloramphenicol
(Chloramphenicol is one of the antibiotics. )

After induction, we added chloramphenicol into the medium and measured optical density
after induction.
 

2. Results
 

2-1 C4HSL-dependent 3OC12HSL production

We measured the expression of GFP in the reporter cell by flow cytometer.

2-2 C4HSL-dependent growth

After induction, optical density were measured to estimate the concentration of the cell.
Fig. 3-1-1
 
Fig. 3-1-2
 

explanation?   

 

3. Materials and methods
 

3-1 Construction

-Strain

All the samples were JM2.300 strain

-Plasmids
 

3-2

3-2-1. C4HSL-dependent 3OC12HSL production assay by using reporter assay

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 37C until the observed OD590 reaches 0.5.
3. Centrifuge 1mL of the sample at 5000g, RT for 1 minute.
4. Suspend the pellet in 1 mL of LB containing Ampicillin（50μg/mL）and Kanamycin(30μg/mL) .
5. Add 30µL of suspension in the following medium.
           Add 30µL of 500µM C12HSL to 3mL LB containing Amp and Kan
           Add 30µL DMSO to 3mL of LB containing Amp and Kan
6. Grow the samples of sender cell at 37°C for 4 hours.
7. Measure optical density every hour.  (If optical density is over 1.0, dilute the cell medium.)
8. Centrifuge sample at 9000g, 4°C for 1minute. Filter sterilize supernatant.
9. Use the supernatant in reporter assay.
 

Reporter Assay
1. Grow the colony of Reporter cell(D～F) in LB containing antibiotic O/N at 37°C.
2. Make a 1:100 dilution in 3 mL of fresh LB+ antibiotic and grow the cells at 37°C until you reach an 0.5 OD590. (fresh culture).
3. Centrifuge sample at 5000g, 25°C RT for 1 minute. Discard the supernatant.
4. Suspended the sample in 3 mL of LB containing Ampicillin(50μg/mL) and Kanamycin(30μg/mL).
5. Add 30µL of suspension in the following medium.
           Filtrate of A①＋3mL of LB containing Amp and Kan
           Filtrate of A②＋3mL of LB containing Amp and Kan
           Filtrate of B①＋3mL of LB containing Amp and Kan
           Filtrate of B②＋3mL of LB containing Amp and Kan
           Filtrate of C①＋3mL of LB containing Amp and Kan
          Filtrate of C②＋3mL of LB containing Amp and Kan
          3OC12HSL＋3mL of LB containing Amp and Kan
          DMSO + 3mL of LB containing Amp and Kan
6. Grow the samples of Reporter cell in incubator at 37°C for 4 hours.
7. Start preparing the flow cytometer 1 h before the end of incubation.
8. Take 200 microL of the sample, and centrifuge at 9000 Xg, 1 min, 4°C.
9. Remove the supernatant by using P1000 pipette.
10. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend.
11. Dispense all of each suspension into a disposable tube through a cell strainer.
12. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).
 

3-2-2. C4HSL-depemdent CmR expression
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 containig antibiotic and grow the cells at 37°C until the observed OD590 reaches 0.5 (fresh culture).
3. Centrifuge 1mL of the sample at 5000g, RT for 1 minute.
4. Suspend the pellet in 1 mL of LB containing Ampicillin（50 microg/mL）and Kanamycin(30 microg/mL)
5. Add 30µL of suspension in the following medium.
           Add 3µL of 5µM C12HSL to 3mL LB containing Amp, Kan(concentration is described upper) and Cm(100 microg /mL).
           Add 3microL DMSO to 3mL of LB containing Amp and Kan.
6. Grow the samples of sender cell at 37°C for 4 hours.
7. Measure optical density every hour.  (If optical density is over 1.0, dilute the cell medium.)
 

4. Reference
 

 

 

Retrieved from "http://2014.igem.org/Team:Tokyo_Tech/Experiment/C4HSL-dependent_3oxoC12HSL_production"