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

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Latest revision as of 03:33, 18 October 2014

Tokyo_Tech

Experiment

Plux and Prhl reporter assay

Contents

1. Introduction

2. Summary of the Experiment

3. Results

4. Materials and Methods

4-1. Construction

4-2. Assay Protocol

5. Reference




1. Introduction

In the modeling (Fig. 3-1-1-1), the better mutualism 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 through the reporter assay (Fig. 3-1-1-2).
Fig. 3-1-1-1. The phase diagram of the intensity of Plux and Prhl promoter	Fig. 3-1-1-2. Plux and Prhl reporter assay flow chart



2. Summary of the Experiment

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

Fig. 3-1-3-1 shows our experimental data of Plux promoter and Prhl promoter. Compared with the modeling results, the expression level of the native BioBrick Prhl promoter (BBa_R0071) was too weak to satisfy our requirement (Fig. 3-1-3-1 lane 2). In other words, the expression of RhlI under the control of Plux promoter (BBa_R0062) was 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. Prepare 2 overnight cultures for each sample in 3 mL LB medium, containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) at 37°C for 12 h. 2. Dilute the overnight cultures to 1 / 100 in fresh LB medium (3 mL) containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) (fresh culture). 3. Incubate the fresh cultures at 37°C until the OD590 reaches 0.3. 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 5. Incubate the samples at 37°C for 4 h. 6. Start preparing the flow cytometer 1 h before the end of incubation. 7. Take 200 microL of the sample, and centrifuge at 9000x g, 1 min, 4°C. 8. Remove the supernatant by using P1000 pipette. 9. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. 10. Dispense all of each suspension into a disposable tube through a cell strainer. 11. Measure the fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).



5. Reference

1. 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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-<meta name="keywords" content="iGEM,2014,Tokyo_Tech,Bank E.coli" />
+<meta name="keywords" content="iGEM,2014,Tokyo_Tech,Bank <i>E.coli</i>" />
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 			        <li><a href="https://2014.igem.org/Team:Tokyo_Tech/Experiment/C4HSL-dependent_3OC12HSL_production" style="width:400px; margin-left:-135px;">C4HSL-dependent 3OC12HSL production</a></li>
 			        <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/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/Symbiosis_confirmation_by_co-culture" style="width:400px; margin-left:-135px;">Mutualism Confirmation ~Co-culture Assay~</a></li>
 			   </ul>
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                      <div align="center"><p class="title-small">Contents</p></div>
                      <p class="info-24"><a href="#Introduction">1. Introduction</a></p>
-                     <p class="info-24"><a href="#Summary">2. Summary of the experiment</a></p>
+                     <p class="info-24"><a href="#Summary">2. Summary of the Experiment</a></p>
                      <p class="info-24"><a href="#Results">3. Results</a></p>
-                     <p class="info-24"><a href="#Materials">4. Materials and methods</a></p>
+                     <p class="info-24"><a href="#Materials">4. Materials and Methods</a></p>
                      <p class="info-18"><a href="#Materials">4-1. Construction</a></p>
                      <p class="info-18"><a href="#Assay">4-2. Assay Protocol</a></p>
@@ Line 95: / Line 95: @@
                </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 through the reporter assay (Fig. 3-1-1-2.). </p></td>
+                 <td colspan="2"><p class="info-18">In the modeling (Fig. 3-1-1-1), the better mutualism 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 through the reporter assay (Fig. 3-1-1-2). </p></td>
                </tr>
                <tr>
@@ Line 102: / Line 102: @@
                    <strong>Fig. 3-1-1-1.</strong> The phase diagram of the intensity of Plux and Prhl promoter    </div></td>
                  <td width="488"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Plux_and.png"><img src="https://static.igem.org/mediawiki/2014/c/cf/Tokyo_Tech_Plux_and.png" width="500" height="190" align="middle" /></a></div>
-                 <div align="center"><strong>Fig. 3-1-1-2.</strong> Plux and Prhl Reporter Assay flow chart</div></td>
+                 <div align="center"><strong>Fig. 3-1-1-2.</strong> Plux and Prhl reporter assay flow chart</div></td>
                </tr>
                <tr>
@@ Line 114: / Line 114: @@
                </tr>
                <tr>
-                 <td colspan="2"><h2>2. Summary of the experiment </h2></td>
+                 <td colspan="2"><h2>2. Summary of the Experiment </h2></td>
                </tr>
                <tr>
@@ Line 120: / Line 120: @@
                </tr>
                <tr>
-                 <td colspan="2" class="info-18"><p class="info-18">Our purpose is to confirm the expression level 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 two plasmids sets shown in below (Fig. 3-1-2-1.). We measured fluorescence intensity by GFP expression when we added signaling molecules.</p>
+                 <td colspan="2" class="info-18"><p class="info-18">Our purpose is to confirm the expression level 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 two plasmids sets shown in below (Fig. 3-1-2-1). We measured the fluorescence intensity by GFP expression when we added signaling molecules.</p>
                    <p class="info-18">We prepared four conditions shown in below.</p>
                    <blockquote>
@@ Line 140: / Line 140: @@
                </tr>
                <tr>
-                 <td colspan="2"><div align="center"><strong>Fig. 3-1-2-1. </strong>Reporter plasmids </div>
+                 <td colspan="2"><div align="center"><strong>Fig. 3-1-2-1. </strong>Reporter Plasmids </div>
                    <div>
                    <div> </div>
@@ Line 172: / Line 172: @@
                </tr>
                <tr>
-                 <td colspan="2"><p class="info-18">From the modeling results, it showed that the expression level of the pre-existing BioBrick Prhl promoter (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>) was too weak to satisfy our requirement (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>) was higher than the expression of LasI under the control of Prhl promoter.
+                 <td colspan="2"><p class="info-18">Fig. 3-1-3-1 shows our experimental data of Plux promoter and Prhl promoter. Compared with the modeling results, the expression level of the native BioBrick Prhl promoter (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>) was too weak to satisfy our requirement (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>) was higher than the expression of LasI under the control of Prhl promoter.
                    </p>
                    <div>
@@ Line 190: / Line 190: @@
                </tr>
                <tr>
-                 <td colspan="2"><h2>4. Materials and methods</h2></td>
+                 <td colspan="2"><h2>4. Materials and Methods</h2></td>
                </tr>
                <tr>
@@ Line 212: / Line 212: @@
                </tr>
                <tr>
-                 <td colspan="2"><p class="info-24">-Plasmids</p></td>
+                 <td colspan="2"><div class="info-18"><p class="info-24">-Plasmids</p> </div></td>
                </tr>
                <tr>
@@ Line 218: / Line 218: @@
                </tr>
                <tr>
-                 <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig3-1-4-1.png"><img src="https://static.igem.org/mediawiki/2014/d/d6/Tokyo_Tech_Fig3-1-4-1.png" width="400" height="122" /></a></div>
+                 <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig3-1-4-1.png"><img src="https://static.igem.org/mediawiki/2014/d/d6/Tokyo_Tech_Fig3-1-4-1.png" width="500"/></a></div>
                </tr>
                <tr>
@@ Line 227: / Line 227: @@
                </tr>
                <tr>
-                 <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig3-1-4-2.png"><img src="https://static.igem.org/mediawiki/2014/3/37/Tokyo_Tech_Fig3-1-4-2.png" width="400" height="123" />                                </a></div>
+                 <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig3-1-4-2.png"><img src="https://static.igem.org/mediawiki/2014/3/37/Tokyo_Tech_Fig3-1-4-2.png" width="500" />                                </a></div>
                </tr>
                <tr>
-                 <td colspan="2">&nbsp;</td>
+                 <td colspan="2"> <div align="center"><strong>Fig. 3-1-4-2.
+&nbsp;</td>
                </tr>
                <tr>
@@ Line 243: / Line 244: @@
                <tr>
                  <td colspan="2"><ol>
-                   <li class="info-18">1.Prepare 2 overnight cultures for each sample  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">1. Prepare 2 overnight cultures for each sample  in 3 mL LB medium, containing ampicillin (50 microg /
+                   mL) and kanamycin (30 microg / mL) at 37°C for 12 h.          </br>    </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). </p>
+                     <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). </p>
                    </li>
                    <li class="info-18">
-                     <p class="info-18">3.Incubate the fresh cultures in 37°C until the OD590 reaches 0.3.</p>
+                     <p class="info-18">3. Incubate the fresh cultures at 37°C until the OD590 reaches 0.3.</p>
                    </li>
-                   <li class="info-18">	Add 30 microL of 500 microM C4HSL, 500 nM 3OC12HSL or DMSO as listed below:                  </li>
+                   <li class="info-18">4. Add 30 microL of 500 microM C4HSL, 500 nM 3OC12HSL or DMSO as listed below:                  </li>
                  </ol>
                    <blockquote>
@@ Line 266: / Line 269: @@
                      </li>
                      <li class="info-18">
-                       <p class="info-18">7.Take 200 microL of the sample, and centrifuge at 9000x g, 1 min, 4°C.</p>
+                       <p class="info-18">7. Take 200 microL of the sample, and centrifuge at 9000x g, 1 min, 4°C.</p>
                      </li>
                      <li class="info-18">
@@ Line 277: / Line 280: @@
                    </ol>
                    <p class="info-18">
-. Measure fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).                                                </p>
+. Measure the fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).                                                </p>
                </tr>
                <tr>