Team:Tokyo Tech/Experiment/Symbiosis confirmation by co-culture

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<h2 class="title">Experiment</h2>
<h2 class="title">Experiment</h2>
                 <span class="meta">Symbiosis confirmation by co-culture</span>
                 <span class="meta">Symbiosis confirmation by co-culture</span>
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<p>Under Construction！</p>
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              <p>&nbsp;</p>
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              <table width="900" border="0">
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                  <td colspan="2"><div align="center" class="title-small">Symbiosis confirmation ~co-culture assay~</div></td>
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<p>&nbsp;</p>
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<div align="center"><img src="http://www.actmp2014.com/images/under_construction%20(1).png" /></div>
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                  <td colspan="2">&nbsp;</td>
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<p>&nbsp;</p>
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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 accomplish symbiosis of the Company cells and the Customer cells, we mixed and co-cultured the two cells.</p>
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                  <p class="info-18">Company&rsquo;s characteristics are 4HSL-dependent survival and 3OSC12HSL production, and Company&rsquo;s characteristics are the opposite from the Customer&rsquo;s. (If you want to know about the cells in more detail, please visit the project page.</p>
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                  <p class="info-18">Each cells&rsquo; functions are confirmed.) These characteristics establish symbiosis between the two cells.</p>
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                  <p class="info-18">This experimentation was focused on confirmation of the symbiosis and its condition. We constructed the Company cells containing GFP and the Customer cells containing RFP. By using the flow cytometer, the symbiosis and the condition become detectable. </p></td>
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                </tr>
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                <tr>
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                  <td colspan="2" class="info-18"><div align="center"><img src="fig3-3-1.png" width="751" height="234" align="middle" /></div></td>
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                  <td colspan="2"><div align="center">Fig. 3-1-2 </div></td>
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                  <td colspan="2"><div align="center"></div></td>
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                  <td colspan="2"><h2>2. Results</h2></td>
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                  <td colspan="2">&nbsp;</td>
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                  <td colspan="2"><p align="center" class="head"><img src="fig3-2-2.png" width="627" height="226" /></p></td>
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                  <td colspan="2"><p align="center"><strong>Fig. 3-3-2. </strong>Growth of the two cells when co-cultured</p></td>
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                  <td colspan="2">&nbsp;</td>
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                </tr>
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                  <td colspan="2"><h2>3.  Materials and methods</h2></td>
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                  <td colspan="2">&nbsp;</td>
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                <tr>
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                  <td colspan="2"><p class="head">3-1  Construction</p></td>
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                </tr>
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                <tr>
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                  <td colspan="2"><p class="head">-Strain</p></td>
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                </tr>
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                <tr>
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                  <td colspan="2"><p class="info-18">All the samples were JM2.300 strain</p></td>
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                </tr>
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                <tr>
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                  <td colspan="2"><p class="head">-Plasmids</p></td>
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                </tr>
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                <tr>
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                  <td colspan="2"><p class="info-18">A. Ptet-GFP-Ptet-RhlR (pSB6A1), Plux-CmR-LasI (pSB3K3) ...Company (type1)</p></td>
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                </tr>
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                <tr>
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                  <td colspan="2"><p class="info-18">B. Ptet-GFP-Ptet-RhiR (pSB6A1), Prhl-CmR-LasI (pSB3K3) ...Company (type2)</p></td>
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                </tr>
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                <tr>
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                  <td colspan="2"><p class="info-18">C. Ptet-GFP-Ptet-RhiR (pSB6A1), Prhl(RL)-CmR-lasI (pSB3K3) ...Company (type3)</p></td>
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                </tr>
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                <tr>
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                  <td colspan="2"><p class="info-18">D. Ptet-RFP-Ptet-LuxR (pSB6A1), Plux-CmR-RhlI (pSB3K3) ...Customer</p></td>
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                </tr>
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                <tr>
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                  <td colspan="2"><p class="info-18">E. Ptet-GFP-Ptet-RhlR (pSB6A1), PlacIq-CmR (pSB3K3) ...Negative control (company)</p></td>
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                </tr>
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                <tr>
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                  <td colspan="2"><p class="info-18">F. Ptet-RFP-Ptet-LuxR (pSB6A1), PlacIq-CmR (pSB3K3) ...Negative control (customer)</p></td>
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                </tr>
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                <tr>
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                  <td colspan="2">&nbsp;</td>
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                </tr>
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                <tr>
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                  <td colspan="2"><p class="head">3-2 Assay Protocol</p></td>
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                </tr>
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                <tr>
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                  <td colspan="2"><ol>
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                    <li 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.</li>
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                  </ol></td>
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                </tr>
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                <tr>
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                  <td colspan="2"><ol>
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                    <li 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].                    </li>
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                  </ol></td>
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                </tr>
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                <tr>
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                  <td colspan="2" class="info-18"><ol>
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                    <li class="info-18">3. Incubate the fresh cultures in 37°C until the observed OD590 reaches 0.5. If the OD becomes over 0.5, dilute to 0.5.</li>
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                  </ol></td>
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                </tr>
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                <tr>
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                  <td colspan="2" class="info-18"><ol>
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                    <li class="info-18">4. Take 1 mL of the sample, and centrifuge at 5000x g, 1 min., 25°C.</li>
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                  </ol></td>
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                </tr>
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                <tr>
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                  <td colspan="2" class="info-18">5. Remove the supernatant.</td>
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                </tr>
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                <tr>
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                  <td colspan="2" class="info-18"><ol>
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                    <li class="info-18">6. Add 1 mL of LB (containing 50 microg / mL ampicilin, 30 microg / mL kanamycin and Cm) and suspend.</li>
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                  </ol></td>
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                </tr>
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                <tr>
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                  <td colspan="2" class="info-18">7. Add the suspension to 3mL of LB medium as listed below.</td>
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                </tr>
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                <tr>
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                  <td colspan="2" class="info-18">LB contains 50 microg / mL ampicilin, 30 microg / mL kanamycin, and Cm.</td>
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                </tr>
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                <tr>
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                  <td width="21" class="info-18">&nbsp;</td>
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                  <td width="869" class="info-18">A 30 microL ＋ D 30 microL</td>
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                </tr>
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                <tr>
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                  <td class="info-18">&nbsp;</td>
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                  <td class="info-18">B 30 microL ＋ D 30 microL</td>
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                </tr>
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                <tr>
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                  <td class="info-18">&nbsp;</td>
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                  <td class="info-18">C 30 microL ＋ D 30 microL</td>
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                </tr>
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                <tr>
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                  <td class="info-18">&nbsp;</td>
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                  <td class="info-18">D 30 microL ＋ E 30 microL</td>
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                </tr>
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                <tr>
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                  <td class="info-18">&nbsp;</td>
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                  <td class="info-18">A 30 microL ＋ F 30 microL</td>
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                </tr>
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                <tr>
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                  <td class="info-18">&nbsp;</td>
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                  <td class="info-18">B 30 microL ＋ F 30 microL</td>
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                </tr>
 +
                <tr>
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                  <td class="info-18">&nbsp;</td>
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                  <td class="info-18">C 30 microL ＋ F 30 microL</td>
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                </tr>
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                <tr>
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                  <td colspan="2" class="info-18"><ol>
 +
                    <li class="info-18">8. Incubate these samples at 37°C for 6 h.</li>
 +
                  </ol></td>
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                </tr>
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                <tr>
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                  <td colspan="2" class="info-18">(During that time, measure the optical density every one hour.)</td>
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                </tr>
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                <tr>
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                  <td colspan="2" class="info-18"><ol>
 +
                    <li class="info-18">9. Measure the fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).</li>
 +
                  </ol></td>
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                </tr>
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                  <td colspan="2">&nbsp;</td>
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                </tr>
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                  <td colspan="2"><h2>4. Reference</h2></td>
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                <tr>
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                  <td colspan="2" class="info-18">&nbsp;</td>
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                </tr>
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              </table>
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Revision as of 15:09, 10 October 2014

Tokyo_Tech

Experiment

Symbiosis confirmation by co-culture

 

Symbiosis confirmation ~co-culture assay~
 
 

1. Summary of the experiment

To accomplish symbiosis of the Company cells and the Customer cells, we mixed and co-cultured the two cells.

Company’s characteristics are 4HSL-dependent survival and 3OSC12HSL production, and Company’s characteristics are the opposite from the Customer’s. (If you want to know about the cells in more detail, please visit the project page.

Each cells’ functions are confirmed.) These characteristics establish symbiosis between the two cells.

This experimentation was focused on confirmation of the symbiosis and its condition. We constructed the Company cells containing GFP and the Customer cells containing RFP. By using the flow cytometer, the symbiosis and the condition become detectable.

Fig. 3-1-2

2. Results
 

Fig. 3-3-2. Growth of the two cells when co-cultured
 

3. Materials and methods
 

3-1 Construction

-Strain

All the samples were JM2.300 strain

-Plasmids

A. Ptet-GFP-Ptet-RhlR (pSB6A1), Plux-CmR-LasI (pSB3K3) ...Company (type1)

B. Ptet-GFP-Ptet-RhiR (pSB6A1), Prhl-CmR-LasI (pSB3K3) ...Company (type2)

C. Ptet-GFP-Ptet-RhiR (pSB6A1), Prhl(RL)-CmR-lasI (pSB3K3) ...Company (type3)

D. Ptet-RFP-Ptet-LuxR (pSB6A1), Plux-CmR-RhlI (pSB3K3) ...Customer

E. Ptet-GFP-Ptet-RhlR (pSB6A1), PlacIq-CmR (pSB3K3) ...Negative control (company)

F. Ptet-RFP-Ptet-LuxR (pSB6A1), PlacIq-CmR (pSB3K3) ...Negative control (customer)
 

3-2 Assay Protocol
  1. 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.
  1. 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].
  1. 3. Incubate the fresh cultures in 37°C until the observed OD590 reaches 0.5. If the OD becomes over 0.5, dilute to 0.5.
  1. 4. Take 1 mL of the sample, and centrifuge at 5000x g, 1 min., 25°C.
5. Remove the supernatant.
  1. 6. Add 1 mL of LB (containing 50 microg / mL ampicilin, 30 microg / mL kanamycin and Cm) and suspend.
7. Add the suspension to 3mL of LB medium as listed below.
LB contains 50 microg / mL ampicilin, 30 microg / mL kanamycin, and Cm.
  A 30 microL ＋ D 30 microL
  B 30 microL ＋ D 30 microL
  C 30 microL ＋ D 30 microL
  D 30 microL ＋ E 30 microL
  A 30 microL ＋ F 30 microL
  B 30 microL ＋ F 30 microL
  C 30 microL ＋ F 30 microL
  1. 8. Incubate these samples at 37°C for 6 h.
(During that time, measure the optical density every one hour.)
  1. 9. Measure the fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).
 

4. Reference
 

 

 

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