Team:Tokyo Tech/Experiment/Plux and Prhl reporter assay

From 2014.igem.org

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<h2 class="title">Experiment</h2>
<h2 class="title">Experiment</h2>
                 <span class="meta">Plux and Prhl reporter assay</span>
                 <span class="meta">Plux and Prhl reporter assay</span>
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<p>Under Construction！</p>
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            <div id="gototop"><a href="#"><img src="https://static.igem.org/mediawiki/2014/5/55/Tokyo_Tech_Go-to-top-icon.png" height="50" /></a></div>
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<p>&nbsp;</p>
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                <td colspan="2"><div align="center" class="title-small">
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<p>&nbsp;</p>
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                  <p class="title-small">Contents</p>
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<p>&nbsp;</p>
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                </div></td>
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<div align="center"><img src="http://www.actmp2014.com/images/under_construction%20(1).png" /></div>
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              </tr>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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                <td colspan="2" class="info-18"><ol>
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<p>&nbsp;</p>
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                  <li>
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<p>&nbsp;</p>
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                    <p class="info-24"><a href="#Introduction">1. Introduction</a></p>
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<p>&nbsp;</p>
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                  </li>
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<p>&nbsp;</p>
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                  <li>
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                    <p class="info-24"><a href="#Summary">2. Summary of the experiment</a></p>
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  </div>
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                  </li>
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    </div>
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                  <li>
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                    <p class="info-24"><a href="#Results">3. Results</a></p>
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  </div>
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                  </li>
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                  <li>                   
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                    <p class="info-24"><a href="#Materials">4. Materials and methods                  </a></p>
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                  </li>
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                </ol>
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                  <ul>
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                    <li> </li>
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                  </ul>
 +
                  <blockquote>
 +
                    <p class="info-24"><a href="#Materials">4-1. Construction</a></p>
 +
                    <p class="info-24"><a href="#Assay">4-2 Assay Protocol</a></p>
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                  </blockquote>
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                  <ol>
 +
                    <li>
 +
                      <p class="info-24"><a href="#Reference">5. Reference</a></p>
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                    </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="entry-long"><p><span class="entry-long" style="clear: both;"><a name="Introduction" id="Introduction"></a></span></p></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2">&nbsp;</td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><h2>1. Introduction</h2></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2">&nbsp;</td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><p class="info-18">In the modeling (Fig. 3-1-1-1.), the better interdependence between Company and Customer requires that the expression of LasI, under the control of Prhl promoter (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>), is the same level as the expression of RhlI, under the control of Plux  promoter (<a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062</a>). Thus, we firstly tested the expression level of Prhl promoter and Plux promoter by the reporter assay (Fig. 3-1-1-2.).</p></td>
 +
              </tr>
 +
              <tr>
 +
                <td width="397"><div align="center">
 +
                  <p><img src="intensity.png" width="276" height="268" /></p>
 +
                  <strong>Fig. 3-1-1-1.</strong> The phase diagram of Plux and Prhl intensity                </div></td>
 +
                <td width="488"><div align="center"><img src="Plux.png" width="500" height="190" align="middle" /></div>
 +
                <div align="center"><strong>Fig. 3-1-1-2.</strong> Plux and Prhl Reporter Assay flow chart</div></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><span class="entry-long" style="clear: both;"><a name="Summary" id="Summary"></a></span></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2">&nbsp;</td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><h2>2. Summary of the experiment </h2></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2">&nbsp;</td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2" class="info-18"><p class="info-18">Our purpose is to confirm actually expression levels of Prhl promoter (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>) and Plux promoter (<a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062</a>). We prepared three plasmids sets shown in below. (Fig. 3-1-2-1.) We measured fluorescence intensity by GFP expression when we added signaling molecules.</p>
 +
                  <p class="info-18">We prepared four conditions as follow.</p>
 +
                  <blockquote>
 +
                    <p class="info-18"> A-1: Culture containing Ptet-LuxR and Plux-GFP cell with 3OC12HSL<br />
 +
                           A-2: Culture containing Ptet-LuxR and Plux-GFP cell with DMSO<br />
 +
                           B-1: Culture containing Ptet-RhlR and Prhl-GFP cell with C4HSL<br />
 +
                         B-2: Culture containing Ptet-RhlR and Prhl-GFP cell with DMSO</p>
 +
                  </blockquote>
 +
                  <div>
 +
                    <div class="info-18"></div>
 +
</div></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><div align="center"><img src="reporter.png" width="400" height="246" />
 +
                </div>
 +
                  <div>
 +
                  <div></div>
 +
                </div></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><div align="center"><strong>Fig. 3-1-2-1. </strong>Reporter plasmids </div>
 +
                  <div>
 +
                  <div> </div>
 +
                    <div> </div>
 +
                    <div> </div>
 +
                </div></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><span class="entry-long" style="clear: both;"><a name="Results" id="Results"></a></span></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2">&nbsp;</td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><h2>3. Results</h2></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2">&nbsp;</td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><div align="center"><img src="plux reporter.png" width="400" height="370" /></div></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><div align="center"><strong>Fig. 3-1-3-1. </strong>Plux and Prhl Reporter Assay result</div></td>
 +
              </tr>       
 +
              <tr>
 +
                <td colspan="2">&nbsp;</td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><p class="info-18">The measured activity of pre-existing Biobrick Prhl promoter (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>) was too weak to satisfy the requirement from our modeling results. (Fig. 3-1-3-1 lane 2) In other words, the expression of RhlI under the control of Plux promoter (<a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062</a>) is higher than the expression of LasI under the control of Prhl promoter.
 +
                  </p>
 +
                  <div>
 +
                    <div> </div>
 +
                    <div> </div>
 +
                    <div></div>
 +
                  </div></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><span class="entry-long" style="clear: both;"><a name="Materials" id="Materials"></a></span></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2">&nbsp;</td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><h2>4. Materials and methods</h2></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2">&nbsp;</td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><p class="head">4-1. Construction</p></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><div class="info-18">
 +
                  <p class="info-24">-Strain</p>
 +
                </div></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><p class="info-18">All the samples were JM2.300 strain
 +
                  </p>
 +
                  <div>
 +
                  <div> </div>
 +
                    <div> </div>
 +
                </div>                  </td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><p class="info-24">-Plasmids</p></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2" class="info-18"><p class="info-18">A. Ptet-LuxR (pSB6A1), Plux-GFP (pSB3K3)</p></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><div align="center"><img src="ptet.png" width="400" height="122" />                                </div>               
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><div align="center"><strong>Fig. 3-1-4-1.                                </strong></div>               
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><p class="info-18">B. Ptet-RhlR (pSB6A1), Prhl-GFP (pSB3K3)</p>                                               
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><div align="center"><img src="psb6a1.png" width="400" height="123" />                                </div>               
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><div align="center"><strong>Fig. 3-1-4-2.                </strong> </div>               
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><span class="entry-long" style="clear: both;"><a name="Assay" id="Assay"></a></span>               
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><p class="head">4-2. Assay Protocol</p>                                              
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><ol>
 +
                  <li class="info-18">1.Prepare 2 overnight cultures of every sample A~D in 3 mL LB medium, containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) at 37°C for 12 h.                  </li>
 +
                  <li class="info-18">
 +
                    <p class="info-18">2. Dilute the overnight cultures to 1 / 100 in fresh LB medium (3 mL) containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) (fresh culture). Make glycerol stocks from the remainders.</p>
 +
                  </li>
 +
                  <li class="info-18">
 +
                    <p class="info-18">3. Incubate the fresh cultures in 37°C until the observed OD590 reaches 0.3 (Actual value 0.42).</p>
 +
                  </li>
 +
                  <li class="info-18">4.                    Add 30 microL of 500 microM C4HSL, 500 nM 3OC12HSL or DMSO as listed below:                  </li>
 +
                </ol>
 +
                  <blockquote>
 +
                    <p class="info-18">   A-1: A + 500 nM 3OC12HSL<br />
 +
                             A-2: A + DMSO<br />
 +
                             B-1: B +500 microM C4HSL<br />
 +
                             B-2: B + DMSO</p>
 +
                  </blockquote>
 +
                  <ol>
 +
                    <li>
 +
                      <p class="info-18">5. Incubate the samples at 37°C for 4 h.</p>
 +
                    </li>
 +
                    <li class="info-18">
 +
                      <p class="info-18">6. Start preparing the flow cytometer 1 h before the end of incubation.</p>
 +
                    </li>
 +
                    <li class="info-18">
 +
                      <p class="info-18">7. Take 200 microL of the sample, and centrifuge at 9000 Xg, 1 min, 4°C.</p>
 +
                    </li>
 +
                    <li class="info-18">
 +
                      <p class="info-18">8. Remove the supernatant by using P1000 pipette.</p>
 +
                    </li>
 +
                    <li class="info-18">
 +
                      <p class="info-18">9. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend.</p>
 +
                    </li>
 +
                    <li class="info-18">10. Dispense all of each suspension into a disposable tube through a cell strainer.                    </li>
 +
                  </ol>
 +
                  <p class="info-18">
 +
                11. Measure fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).                                                </p>               
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><span class="entry-long" style="clear: both;"><a name="Reference" id="Reference"></a></span>                               
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2">               
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><h2>5. Reference</h2></td>
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2">               
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"><p class="info-18">1. Kendall M. Gray <em>et al. </em>(1994) Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa. Journal of Bacteriology 176(10): 3076–3080</p>                                               
 +
              </tr>
 +
              <tr>
 +
                <td colspan="2"></tr>
 +
            </table>
 +
            </div>
 +
            </div>
 +
  </div>
<!-- end #content -->
<!-- end #content -->

Revision as of 02:40, 17 October 2014

Tokyo_Tech

Experiment

Plux and Prhl reporter assay

Contents

  1. 1. Introduction

  2. 2. Summary of the experiment

  3. 3. Results

  4. 4. Materials and methods

4-1. Construction

4-2 Assay Protocol

  1. 5. Reference

 

1. Introduction
 

In the modeling (Fig. 3-1-1-1.), the better interdependence between Company and Customer requires that the expression of LasI, under the control of Prhl promoter (BBa_R0071), is the same level as the expression of RhlI, under the control of Plux  promoter (BBa_R0062). Thus, we firstly tested the expression level of Prhl promoter and Plux promoter by the reporter assay (Fig. 3-1-1-2.).

Fig. 3-1-1-1. The phase diagram of Plux and Prhl intensity
Fig. 3-1-1-2. Plux and Prhl Reporter Assay flow chart
 

2. Summary of the experiment

 

Our purpose is to confirm actually expression levels of Prhl promoter (BBa_R0071) and Plux promoter (BBa_R0062). We prepared three plasmids sets shown in below. (Fig. 3-1-2-1.) We measured fluorescence intensity by GFP expression when we added signaling molecules.

We prepared four conditions as follow.

A-1: Culture containing Ptet-LuxR and Plux-GFP cell with 3OC12HSL
     A-2: Culture containing Ptet-LuxR and Plux-GFP cell with DMSO
     B-1: Culture containing Ptet-RhlR and Prhl-GFP cell with C4HSL
     B-2: Culture containing Ptet-RhlR and Prhl-GFP cell with DMSO
Fig. 3-1-2-1. Reporter plasmids
 

3. Results
 
Fig. 3-1-3-1. Plux and Prhl Reporter Assay result
 

The measured activity of pre-existing Biobrick Prhl promoter (BBa_R0071) was too weak to satisfy the requirement from our modeling results. (Fig. 3-1-3-1 lane 2) In other words, the expression of RhlI under the control of Plux promoter (BBa_R0062) is higher than the expression of LasI under the control of Prhl promoter.

 

4. Materials and methods
 

4-1. Construction

-Strain

All the samples were JM2.300 strain

-Plasmids

A. Ptet-LuxR (pSB6A1), Plux-GFP (pSB3K3)
Fig. 3-1-4-1.

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

Fig. 3-1-4-2.

4-2. Assay Protocol

  1. 1.Prepare 2 overnight cultures of every sample A~D in 3 mL LB medium, containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) at 37°C for 12 h.

  2. 2. Dilute the overnight cultures to 1 / 100 in fresh LB medium (3 mL) containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) (fresh culture). Make glycerol stocks from the remainders.

  3. 3. Incubate the fresh cultures in 37°C until the observed OD590 reaches 0.3 (Actual value 0.42).

  4. 4. Add 30 microL of 500 microM C4HSL, 500 nM 3OC12HSL or DMSO as listed below:

   A-1: A + 500 nM 3OC12HSL
       A-2: A + DMSO
       B-1: B +500 microM C4HSL
       B-2: B + DMSO

  1. 5. Incubate the samples at 37°C for 4 h.

  2. 6. Start preparing the flow cytometer 1 h before the end of incubation.

  3. 7. Take 200 microL of the sample, and centrifuge at 9000 Xg, 1 min, 4°C.

  4. 8. Remove the supernatant by using P1000 pipette.

  5. 9. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend.

  6. 10. Dispense all of each suspension into a disposable tube through a cell strainer.

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

5. Reference

1. Kendall M. Gray et al. (1994) Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa. Journal of Bacteriology 176(10): 3076–3080

 

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