Team:Tokyo Tech/Experiment/Prhl reporter assay

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Latest revision as of 03:34, 18 October 2014

Tokyo_Tech

Experiment

Improved Prhl reporter assay

Contents
1. Summary of the Experiment 2. Results 3. Replication 4. Materials and Methods 4-1. Construction 4-2. Assay Protocol 5. Reference




1. Summary of the experiment
We added three improved C4HSL-dependent promoters with high maximum expression level by combinations of regulatory-protein binding sites. (Fig. 3-2-1-1.)
First, we designed an improved Lux promoter which has two RhlR binding sites instead of two LuxR binding sites (Prhl(RR): BBa_K1529320) , as tried in a previous paper (Chuang 2009). To evaluate the function of this promoter, we constructed Prhl(RR)-GFP (BBa_K1529321) plasmids and measured the fluorescence intensity by flow cytometer. In the measurement, we confirmed that GFP under the control of Prhl(RR) promoter showed about 20-fold higher in the fluorescence than that of the native Prhl promoter (BBa_R0071).
However, Prhl(RR) promoter showed a significant leak in the absence of C4HSL. High level of leak is not suitable for the Company-Customer relationship because their mutualism will be broken.
In order to lessen the leak and increase the maximum expression level, we newly designed two promoters, Prhl(LR) (BBa_K1529310) and Prhl(RL) (BBa_K1529300). These promoters have one LuxR binding site and one RhlR binding site. We changed either the former RhlR binding site of Prhl(RR) promoter to LuxR binding site (Prhl(LR)) or the latter RhlR binding site to Lux binding site (Prhl(RL)).
One of our new promoter, Prhl(RL) improved in its expression level while keeping the low leak. GFP under the control of Prhl(RL) promoter showed about 7-fold higher in the fluorescence than that of the native Prhl promoter. The leak was no more than 2-fold high.
Although the other Prhl(LR) promoter showed a higher maximum expression level, it showed a significant leak like Prhl(RR) promoter. GFP under the control of Prhl(LR) promoter showed about 7-fold higher in the fluorescence than that of the original Prhl promoter. However, the leak showed no less than 25-fold high. Thus we used our improved Prhl(RL) (BBa_K1529300) in the following experiments and modelings.


Fig. 3-2-1-1. The design of our Prhl promoters




2. Results
We measured the GFP expression with the four different promoters (Prhl (BBa_R0071), Prhl(RR) (BBa_K1529320), Prhl(LR) (BBa_K1529310), and Prhl(RL) (BBa_K1529300)) by flow cytometer(Fig. 3-2-2-1). Each promoter was tested in the presence and also in the absence of C4HSL (See Materials and Methods for detailed procedures).


Fig. 3-2-2-1. The four promoters we have tested

Fig. 3-2-2-2 shows the fluorescence intensity detected by flow cytometer. Fig. 3-2-2-3 is the extracted data which shows the comparison of the three promoters: Prhl, Prhl(RR), and Prhl(RL).
As Fig. 3-2-2-2 shows, when induced by C4HSL, Prhl(RR) promoter showed higher maximum expression level and higher leak than the native Prhl promoter.
Although Prhl(RL) promoter had lower maximum expression level compared to Prhl(RR) promoter, it had the highest induced/not-induced ratio. This means Prhl(RL) promoter has little leak. Therefore, we can say that Prhl(RL) promoter is the best improved Prhl promoter due to the advantages of less leak and higher expression level.


Fig. 3-2-2-2. The fluorescence intensity of the cells (with positive and negative controls)



Fig. 3-2-2-3. The fluorescence intensity of the cells with the native Prhl (BBa_R0071), Prhl(RR) (BBa_K1529320), and Prhl(RL) (BBa_K1529300) promoter




3. Replication
To confirm the intensity of Prhl(RL) promoter, we did replication study of the reporter assay.
We prepared three plasmids shown below and measured the fluorescence intensity by GFP expression when we added the signaling molecules. Detail of the protocol of this study is written in the Plux and Prhl promoter assay page.
- Ptet-LuxR(6A1), Plux-GFP(3K3)
- Ptet-RhlR(6A1), Prhl-GFP(3K3)
- Ptet-RhlR(6A1), Prhl(RL)-GFP(3K3)


Fig. 3-2-3-1. The fluorescence intensity of the cells with Plux, Prhl and Prhl(RL) promoter.
Fig. 3-2-3-1 shows that Prhl(RL) promoter is still weaker than Plux promoter. Prhl(RL) promoter does not have enough power for our project, so we have to improve Prhl promoter further.



4. Materials and methods
4-1. Construction

-Strain
All the samples were JM2.300 strain

-Plasmids
A. Ptet-RhlR (pSB6A1), Prhl-GFP (pSB3K3)


Fig. 3-2-4-1.

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


Fig. 3-2-4-2.

C. Ptet-RhlR (pSB6A1)　Prhl(LR)GFP (pSB3K3)


Fig. 3-2-4-3.

D. Ptet-RhlR (pSB6A1)　Prhl(RL)-GFP (pSB3K3)


Fig. 3-2-4-4.

E. Ptet-RhlR (pSB6A1)　PlacUV5-GFP (pSB3K3) ...Positive control


Fig. 3-2-4-5.

F. Ptet-RhlR (pSB6A1)　promoter less-GFP(pSB3K3) ...Negative control


Fig. 3-2-4-6.

4-2. Assay Protocol

1. Prepare 2 overnight cultures for each sample A~F 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 in 37°C until the OD590 reaches 0.3.
4. Add 30 microL of 500 microM C4HSL or DMSO as listed below:
A-5 microM: A + C4HSL
A-0 microM: A + DMSO
B-5 microM: B + C4HSL
B-0 microM: B + DMSO
C-5 microM: C + C4HSL
C-0 microM: C + DMSO
D-5 microM: D + C4HSL
D-0 microM: D + DMSO
E-5 microM: E + C4HSL
E-0 microM: E + DMSO
F-5 microM: F + C4HSL
F-0 microM: F + 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 fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).



5. Reference
John S. Chuang et al. (2009) Simpson’s Paradox in a Synthetic Microbial System. SCIENCE 323: 272-275

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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>
 			</li>
-			<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Modeling">Modeling</a>
+			<li><a href="#">Modeling</a>
                             <ul>
                                 <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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          <div class="post">
 		<h2 class="title">Experiment</h2>
-                 <span class="meta">Prhl reporter assay</span>
+                 <span class="meta">Improved Prhl reporter assay</span>
 	    <div class="entry-long">
            <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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                     <td colspan="2"><ol>
                       <li>
-                        <p class="info-24"><a href="#1">1. Summary of the experiment</a></p>
+                        <p class="info-24"><a href="#1">1. Summary of the Experiment</a></p>
                       </li>
                       <li>                     </li>
                       <li>
                         <p class="info-24"><a href="#2">2. Results</a></p>
+                     </li>
+                      <li>
+                       <p class="info-24"><a href="#3">3. Replication </a></p>
+                     </li>
+                     <li>
+                       <p class="info-24"><a href="#4">4. Materials and Methods </a></p>
+                     </li>
+                     <li>
+                       <p class="info-18"><a href="#4-1">4-1. Construction</a></p>
+                     </li>
+                     <li>
+                       <p class="info-18"><a href="#4-2">4-2. Assay Protocol</a></p>
                       </li>
                       <li>
-                        <p class="info-24"><a href="#3">3. Materials and methods </a></p>
+                        <p class="info-24"><a href="#5">5. Reference </a></p>
                       </li>
                     </ol></td>
@@ Line 105: / Line 117: @@
                   </tr>
                   <tr>
-                    <td colspan="2" class="info-18"><p class="info-18">Prhl promoter is a regulatory part activated by RhlR in the presence of N-butyryl-homoserine lactone (also known as C4HSL). Existing Prhl promoter (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>) has a low expression level even when it is activated.
+                    <td colspan="2" class="info-18"><p class="info-18">We added three improved C4HSL-dependent promoters with high maximum expression level by combinations of regulatory-protein binding sites. (Fig. 3-2-1-1.)
                       </p>
                       <div>
@@ Line 112: / Line 124: @@
                   </tr>
                   <tr>
-                    <td colspan="2"><p class="info-18">In order to improve this expression level, we designed a new Lux promoter which has two RhlR binding sites instead of two LuxR binding sites (Prhl(RR): <a href="http://parts.igem.org/Part:BBa_K1529320">BBa_K1529320</a>). </p>
+                    <td colspan="2"><p class="info-18">First, we designed an improved Lux promoter which has two RhlR binding sites instead of two LuxR binding sites (Prhl(RR):<a href="http://parts.igem.org/Part:BBa_K1529320"> BBa_K1529320</a>) , as tried in a previous paper (Chuang 2009). To evaluate the function of this promoter, we constructed Prhl(RR)-GFP (<a href="http://parts.igem.org/Part:BBa_K1529321">BBa_K1529321</a>) plasmids and measured the fluorescence intensity by flow cytometer. In the measurement, we confirmed that GFP under the control of Prhl(RR) promoter showed about 20-fold higher in the fluorescence than that of the native Prhl promoter (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>). </p>
                       <div>
                         <div></div>
@@ Line 118: / Line 130: @@
                   </tr>
                   <tr>
-                    <td colspan="2"><p class="info-18">To evaluate the function of this promoter, we constructed Prhl(RR)-GFP plasmids and measured the fluorescence intensity by flow cytometer. In the measurement, we confirmed that GFP under the control of Prhl(RR) promoter showed about 20-fold higher in the fluorescence than that of the original Prhl promoter (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>) (See <strong>2. Results</strong>). </p>
+                    <td colspan="2"><p class="info-18">However, Prhl(RR) promoter showed a significant leak in the absence of C4HSL. High level of leak is not suitable for the Company-Customer relationship because their mutualism will be broken. </p>
                       <div>
                         <div> </div>
@@ Line 126: / Line 138: @@
                   </tr>
                   <tr>
-                    <td colspan="2"><p class="info-18">However, our Prhl(RR) promoter showed a significant leak in the absence of C4HSL(See 2. Results). In order to lessen the leak and increase the maximum expression level, we newly designed two promoters, Prhl(LR) (<a href="http://parts.igem.org/Part:BBa_K1529310">BBa_K1529310</a>) and Prhl(RL) (<a href="http://parts.igem.org/Part:BBa_K1529300">BBa_K1529300</a>). These promoters have one LuxR binding site and one RhlR binding site. We changed either the upper RhlR binding site of Prhl(RR) promoter to LuxR binding site (Prhl(LR)) or the latter RhlR binding site to Lux binding site (Prhl(RL)). </p>
+                    <td colspan="2"><p class="info-18">In order to lessen the leak and increase the maximum expression level, we newly designed two promoters, Prhl(LR) (<a href="http://parts.igem.org/Part:BBa_K1529310">BBa_K1529310</a>) and Prhl(RL) (<a href="http://parts.igem.org/Part:BBa_K1529300">BBa_K1529300</a>). These promoters have one LuxR binding site and one RhlR binding site. We changed either the former RhlR binding site of Prhl(RR) promoter to LuxR binding site (Prhl(LR)) or the latter RhlR binding site to Lux binding site (Prhl(RL)). </p>
                       <div>
                         <div> </div>
@@ Line 135: / Line 147: @@
                   </tr>
                   <tr>
-                    <td colspan="2"><p class="info-18">Then, we inserted these promoters to the upstream of GFP coding sequence and measured the fluorescence intensity. Prhl(LR) promoter showed a higher maximum expression level, but also showed a significant leak like Prhl(RR) promoter. On the other hand, Prhl(RL) promoter had less leak while keeping the high expression level (See <strong>2. Results</strong>). </p>
+                    <td colspan="2"><p class="info-18">One of our new promoter, Prhl(RL) improved in its expression level while keeping the low leak. GFP under the control of Prhl(RL) promoter showed about 7-fold higher in the fluorescence than that of the native Prhl promoter. The leak was no more than 2-fold high. </p>
                       <div>
                         <div> </div>
@@ Line 143: / Line 155: @@
                     </div></td>
                   </tr>
+                 <tr>
+                   <td colspan="2"><p class="info-18">Although the other Prhl(LR) promoter showed a higher maximum expression level, it showed a significant leak like Prhl(RR) promoter. GFP under the control of Prhl(LR) promoter showed about 7-fold higher in the fluorescence than that of the original Prhl promoter. However, the leak showed no less than 25-fold high. Thus we used our improved Prhl(RL) (<a href="http://parts.igem.org/Part:BBa_K1529300">BBa_K1529300</a>) in the following experiments and modelings. </p>
+                     <div>
+                       <div> </div>
+                       <div> </div>
+                       <div> </div>
+                       <div> </div>
+                   </div></td>
+                 </tr>
                   <tr>
                     <td colspan="2">&nbsp;</td>
                   </tr>
                   <tr>
-                    <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-1.png"><img src="https://static.igem.org/mediawiki/2014/thumb/d/d5/Tokyo_Tech_Fig._3-4-1.png/800px-Tokyo_Tech_Fig._3-4-1.png" alt="" width="450" /></a></div></td>
+                    <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Designs_of_tmproved_Prhl.jpg"><img src="https://static.igem.org/mediawiki/2014/thumb/2/2d/Tokyo_Tech_Designs_of_tmproved_Prhl.jpg/800px-Tokyo_Tech_Designs_of_tmproved_Prhl.jpg" alt="" width="650" /></a></div></td>
                   </tr>
                   <tr>
-                    <td colspan="2"><div align="center">Fig. 3-4-1. The design of our Rhl promoters</div></td>
+                    <td colspan="2"><div align="center"><strong>Fig. 3-2-1-1.</strong> The design of our Prhl promoters</div></td>
                   </tr>
                   <tr>
@@ Line 168: / Line 190: @@
                   </tr>
                   <tr>
-                    <td colspan="2"><p class="info-18">We measured the GFP expression with the four different promoters (Prhl (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>), Prhl(RR) (<a href="http://parts.igem.org/Part:BBa_K1529320">BBa_K1529320</a>), Prhl(LR) (<a href="http://parts.igem.org/Part:BBa_K1529310">BBa_K1529310</a>), and Prhl(RL) (<a href="http://parts.igem.org/Part:BBa_K1529300">BBa_K1529300</a>)) by flow cytometer. Each promoter was tested in the presence and also in the absence of C4HSL (See Materials and Methods for detailed procedures) .</p>
+                    <td colspan="2"><p class="info-18">We measured the GFP expression with the four different promoters (Prhl (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>), Prhl(RR) (<a href="http://parts.igem.org/Part:BBa_K1529320">BBa_K1529320</a>), Prhl(LR) (<a href="http://parts.igem.org/Part:BBa_K1529310">BBa_K1529310</a>), and Prhl(RL) (<a href="http://parts.igem.org/Part:BBa_K1529300">BBa_K1529300</a>)) by flow cytometer(Fig. 3-2-2-1). Each promoter was tested in the presence and also in the absence of C4HSL (See Materials and Methods for detailed procedures).</p>
                       <div>
                         <div> </div>
@@ Line 182: / Line 204: @@
                   </tr>
                   <tr>
-                    <td colspan="2"><div align="center">Fig. 3-4-2. The four promoters we tested</div></td>
+                    <td colspan="2"><div align="center"><strong>Fig. 3-2-2-1.</strong> The four promoters we have tested</div></td>
                   </tr>
                   <tr>
@@ Line 188: / Line 210: @@
                   </tr>
                   <tr>
-                    <td colspan="2"><p class="info-18">Fig. 3-4-3 shows the fluorescence intensity detected by flow cytometer. Fig. 3-4-4 is the extracted data which shows the comparison of the promoters: Prhl, Prhl(RR), and Prhl(RL).</p>
+                    <td colspan="2"><p class="info-18">Fig. 3-2-2-2 shows the fluorescence intensity detected by flow cytometer. Fig. 3-2-2-3 is the extracted data which shows the comparison of the three promoters: Prhl, Prhl(RR), and Prhl(RL).</p>
                       <div>
                         <div class="info-18"></div>
@@ Line 194: / Line 216: @@
                   </tr>
                   <tr>
-                    <td colspan="2"><p class="info-18">As Fig. 3-4-4 shows, when induced by C4HSL, Prhl(RR) promoter showed higher maximum expression level and higher leak than the original Prhl promoter. </p>
+                    <td colspan="2"><p class="info-18">As Fig. 3-2-2-2 shows, when induced by C4HSL, Prhl(RR) promoter showed higher maximum expression level and higher leak than the native Prhl promoter. </p>
                       <div>
                         <div> </div>
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                   </tr>
                   <tr>
-                    <td><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-3.png"><img src="https://static.igem.org/mediawiki/2014/thumb/2/26/Tokyo_Tech_Fig._3-4-3.png/800px-Tokyo_Tech_Fig._3-4-3.png" alt="" width="400" /></a></td>
+                    <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-3.png"><img src="https://static.igem.org/mediawiki/2014/thumb/2/26/Tokyo_Tech_Fig._3-4-3.png/800px-Tokyo_Tech_Fig._3-4-3.png" alt="" width="750" /></a></div></td>
-                   <td><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-4.png"><img src="https://static.igem.org/mediawiki/2014/thumb/6/6e/Tokyo_Tech_Fig._3-4-4.png/800px-Tokyo_Tech_Fig._3-4-4.png" alt="" width="400" /></a></td>
                   </tr>
                   <tr>
-                    <td><div align="center">Fig. 3-4-3. The Fluorescence intensity of the cells  <br />
+                    <td colspan="2"><div align="center"><strong>Fig. 3-2-2-2.</strong> The fluorescence intensity of the cells (with positive and negative controls)</div></td>
-                   (with positive and negative controls)</div></td>
-                   <td><div align="center">Fig. 3-4-4. The fluorescence intensity of the cells with the original Prhl (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>), Prhl(RR) (<a href="http://parts.igem.org/Part:BBa_K1529320">BBa_K1529320</a>), and Prhl(RL) (<a href="http://parts.igem.org/Part:BBa_K1529300">BBa_K1529300</a>)
-                       <div>
-                         <div> </div>
-                       </div>
-                   </div></td>
                   </tr>
                   <tr>
                     <td>&nbsp;</td>
                     <td>&nbsp;</td>
+                 </tr>
+                 <tr>
+                   <td>&nbsp;</td>
+                   <td>&nbsp;</td>
+                 </tr>
+                 <tr>
+                  <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-4.png"><img src="https://static.igem.org/mediawiki/2014/6/6e/Tokyo_Tech_Fig._3-4-4.png" alt="" width="400" /></a></div></td>
+                 </tr>
+                 <tr>
+                   <td colspan="2"><div align="center"><strong>Fig. 3-2-2-3.</strong> The fluorescence intensity of the cells with the native Prhl (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>), Prhl(RR) (<a href="http://parts.igem.org/Part:BBa_K1529320">BBa_K1529320</a>), and Prhl(RL) (<a href="http://parts.igem.org/Part:BBa_K1529300">BBa_K1529300</a>) promoter</td>
+                 </tr>
+                 <tr>
+                   <td>&nbsp;</td>
+                   <td>&nbsp;</td>
+                 </tr>
+                 <tr>
+                   <td colspan="2">&nbsp;</td>
+                 </tr>
+                 <tr>
+                   <td colspan="2" class="entry-long"><a name="3" id="3"></a></td>
+                 </tr>
+                 <tr>
+                   <td colspan="2">&nbsp;</td>
+                 </tr>
+                 <tr>
+                   <td colspan="2"><h2>3. Replication</h2></td>
+                 </tr>
+                 <tr>
+                   <td colspan="2"><p class="info-18">To confirm the intensity of Prhl(RL) promoter, we did replication study of the reporter assay.</p></td>
+                 </tr>
+                 <tr>
+                   <td colspan="2"><p class="info-18">We prepared three plasmids shown below and measured the fluorescence intensity by GFP expression when we added the signaling molecules. Detail of the protocol of this study is written in the <a href="https://2014.igem.org/Team:Tokyo_Tech/Experiment/Plux_and_Prhl_reporter_assay">Plux and Prhl promoter assay page.</a></p></td>
+                 </tr>
+                 <tr>
+                   <td colspan="2"><p class="info-18">- Ptet-LuxR(6A1), Plux-GFP(3K3)</p></td>
+                 </tr>
+                 <tr>
+                   <td colspan="2"><p class="info-18">- Ptet-RhlR(6A1), Prhl-GFP(3K3)</p></td>
+                 </tr>
+                 <tr>
+                   <td colspan="2"><p class="info-18">- Ptet-RhlR(6A1), Prhl(RL)-GFP(3K3)</p></td>
+                 </tr>
+                 <tr>
+                   <td>&nbsp;</td>
+                   <td>&nbsp;</td>
+                 </tr>
+                 <tr>
+                   <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Plux_Prhl_Reporter_Assay_Replication_Result.png"><img src="https://static.igem.org/mediawiki/2014/2/2f/Tokyo_Tech_Plux_Prhl_Reporter_Assay_Replication_Result.png" width="500"/></a></div></td>
+                 </tr>
+                 <tr>
+                   <td colspan="2"><div align="center"><strong>Fig. 3-2-3-1.</strong> The fluorescence intensity of the cells with Plux, Prhl and Prhl(RL) promoter.</div></td>
+                 </tr>
+                 <tr>
+                 <td colspan="2"><p class="info-18">Fig. 3-2-3-1 shows that Prhl(RL) promoter is still weaker than Plux promoter. Prhl(RL) promoter does not have enough power for our project, so we have to improve Prhl promoter further.</p></td>
                   </tr>
                   <tr>
@@ Line 242: / Line 311: @@
                   </tr>
                   <tr>
-                    <td colspan="2" class="entry-long"><span class="entry-long" style="clear: both;"><a name="3" id="3"></a></span></td>
+                    <td colspan="2" class="entry-long"><span class="entry-long" style="clear: both;"><a name="4" id="4"></a></span></td>
                   </tr>
                   <tr>
@@ Line 248: / Line 317: @@
                   </tr>
                   <tr>
-                    <td colspan="2"><h2>3. Materials and methods</h2></td>
+                    <td colspan="2"><h2>4. Materials and methods <a name="4-1" id="4-1"></a></h2></td>
                   </tr>
                   <tr>
-                    <td colspan="2" class="head">3-1. Construction</td>
+                    <td colspan="2" class="head">4-1. Construction</td>
                   </tr>
                   <tr>
@@ Line 277: / Line 346: @@
                     <td colspan="2"><div align="center">
                       <blockquote>
-                        <p><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-5.png"><img src="https://static.igem.org/mediawiki/2014/2/27/Tokyo_Tech_Fig._3-4-5.png" alt="" width="600" /></a></p>
+                        <p><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-5.png"><img src="https://static.igem.org/mediawiki/2014/2/27/Tokyo_Tech_Fig._3-4-5.png" alt="" width="500" /></a></p>
                       </blockquote>
                     </div></td>
                   </tr>
                   <tr>
-                    <td colspan="2"><div align="center">Fig. 3-4-5. </div></td>
+                    <td colspan="2"><div align="center"><strong>Fig. 3-2-4-1.</strong> </div></td>
                   </tr>
                   <tr>
@@ Line 294: / Line 363: @@
                   </tr>
                   <tr>
-                    <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-6.png"><img src="https://static.igem.org/mediawiki/2014/1/10/Tokyo_Tech_Fig._3-4-6.png" alt="" width="600" /></a></div></td>
+                    <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-6.png"><img src="https://static.igem.org/mediawiki/2014/1/10/Tokyo_Tech_Fig._3-4-6.png" alt="" width="500" /></a></div></td>
                   </tr>
                   <tr>
-                    <td colspan="2"><div align="center">Fig. 3-4-6. </div></td>
+                    <td colspan="2"><div align="center"><strong>Fig. 3-2-4-2.</strong> </div></td>
                   </tr>
                   <tr>
@@ Line 309: / Line 378: @@
                   </tr>
                   <tr>
-                    <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-7.png"><img src="https://static.igem.org/mediawiki/2014/5/50/Tokyo_Tech_Fig._3-4-7.png" alt="" width="600" /></div></td>
+                    <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-7.png"><img src="https://static.igem.org/mediawiki/2014/5/50/Tokyo_Tech_Fig._3-4-7.png" alt="" width="500" /></div></td>
                   </tr>
                   <tr>
-                    <td colspan="2"><div align="center">Fig. 3-4-7.</div></td>
+                    <td colspan="2"><div align="center"><strong>Fig. 3-2-4-3.</strong></div></td>
                   </tr>
                   <tr>
@@ Line 324: / Line 393: @@
                   </tr>
                   <tr>
-                    <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-8.png"><img src="https://static.igem.org/mediawiki/2014/9/98/Tokyo_Tech_Fig._3-4-8.png" alt="" width="600" /></a></div></td>
+                    <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-8.png"><img src="https://static.igem.org/mediawiki/2014/9/98/Tokyo_Tech_Fig._3-4-8.png" alt="" width="500" /></a></div></td>
                   </tr>
                   <tr>
-                    <td colspan="2"><div align="center">Fig. 3-4-8.</div></td>
+                    <td colspan="2"><div align="center"><strong>Fig. 3-2-4-4.</strong></div></td>
                   </tr>
                   <tr>
@@ Line 339: / Line 408: @@
                   </tr>
                   <tr>
-                    <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-9.png"><img src="https://static.igem.org/mediawiki/2014/1/1a/Tokyo_Tech_Fig._3-4-9.png" alt="" width="600" /></a></div></td>
+                    <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-9.png"><img src="https://static.igem.org/mediawiki/2014/1/1a/Tokyo_Tech_Fig._3-4-9.png" alt="" width="500" /></a></div></td>
                   </tr>
                   <tr>
-                    <td colspan="2"><div align="center">Fig. 3-4-9.</div></td>
+                    <td colspan="2"><div align="center"><strong>Fig. 3-2-4-5.</strong></div></td>
                   </tr>
                   <tr>
@@ Line 354: / Line 423: @@
                   </tr>
                   <tr>
-                    <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-10.png"><img src="https://static.igem.org/mediawiki/2014/0/0a/Tokyo_Tech_Fig._3-4-10.png" alt="" width="600" /></a></div></td>
+                    <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-10.png"><img src="https://static.igem.org/mediawiki/2014/0/0a/Tokyo_Tech_Fig._3-4-10.png" alt="" width="500" /></a></div></td>
                   </tr>
                   <tr>
-                    <td colspan="2"><div align="center">Fig. 3-4-10.</div></td>
+                    <td colspan="2"><div align="center"><strong>Fig. 3-2-4-6.</strong></div></td>
                   </tr>
                   <tr>
-                    <td colspan="2">&nbsp;</td>
+                    <td colspan="2">&nbsp;<a name="3-2" id="3-2"></a></td>
                   </tr>
                   <tr>
-                    <td colspan="2" class="head">3-2. Assay Protocol</td>
+                    <td colspan="2" class="head">4-2. Assay Protocol</td>
                   </tr>
                   <tr>
@@ Line 369: / Line 438: @@
                   </tr>
                   <tr>
-                    <td colspan="2"><p 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.</p></td>
+                    <td colspan="2" class="info-18">1. Prepare  2 overnight cultures for each sample A~F in 3 mL LB medium, containing  ampicillin (50 microg /mL) and kanamycin (30 microg / mL) at 37°C for 12 h.</td>
                   </tr>
                   <tr>
-                    <td colspan="2"><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>                     </td>
+                    <td colspan="2" 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).                      </td>
                   </tr>
                   <tr>
-                    <td colspan="2"><p class="info-18">3. Incubate the fresh cultures in 37°C until the observed OD590  reaches 0.3 (Actual value 0.42).</p></td>
+                    <td colspan="2" class="info-18">3. Incubate the fresh cultures in 37°C until the OD590  reaches 0.3.</td>
                   </tr>
                   <tr>
-                    <td colspan="2"><p class="info-18">4. Add 30 microL of 500 microM C4HSL or DMSO as listed below:</p>                   </td>
+                    <td colspan="2" class="info-18">4. Add 30 microL of 500 microM C4HSL or DMSO as listed below:             </td>
                   </tr>
                   <tr>
-                    <td colspan="2"><blockquote class="info-18">                     A-5 μM: A + C4HSL</blockquote></td>
+                    <td colspan="2" class="info-18" style="text-indent:50px;"> A-5 microM: A + C4HSL</blockquote></td>
                   </tr>
                   <tr>
-                    <td colspan="2"><blockquote class="info-18">                     A-0 μM: A + DMSO</blockquote></td>
+                    <td colspan="2" class="info-18" style="text-indent:50px;">A-0 microM: A + DMSO</blockquote></td>
                   </tr>
                   <tr>
-                    <td colspan="2"><blockquote class="info-18">B-5  μM: B + C4HSL</blockquote></td>
+                    <td colspan="2" class="info-18" style="text-indent:50px;">B-5 microM: B + C4HSL</blockquote></td>
                   </tr>
                   <tr>
-                    <td colspan="2"><blockquote class="info-18">B-0  μM: B + DMSO</blockquote></td>
+                    <td colspan="2" class="info-18" style="text-indent:50px;">B-0 microM: B + DMSO</blockquote></td>
                   </tr>
                   <tr>
-                    <td colspan="2"><blockquote class="info-18">C-5  μM: C + C4HSL</blockquote></td>
+                    <td colspan="2" class="info-18" style="text-indent:50px;">C-5 microM: C + C4HSL</blockquote></td>
                   </tr>
                   <tr>
-                    <td colspan="2"><td>
+                    <td colspan="2" class="info-18" style="text-indent:50px;">C-0 microM: C + DMSO</blockquote></td>
                   </tr>
+                 <tr>
+                   <td colspan="2" class="info-18" style="text-indent:50px;">D-5 microM: D + C4HSL</blockquote></td>
+                 </tr>
+                 <tr>
+                   <td colspan="2" class="info-18" style="text-indent:50px;">D-0 microM: D + DMSO</blockquote></td>
+                 </tr>
+                 <tr>
+                  <td colspan="2" class="info-18" style="text-indent:50px;">E-5 microM: E + C4HSL</blockquote></td>
+                 </tr>
+                 <td colspan="2" class="info-18" style="text-indent:50px;">E-0 microM: E + DMSO</blockquote></td>
+                 </tr>
+                 <tr>
+                   <td colspan="2" class="info-18" style="text-indent:50px;">F-5 microM: F + C4HSL</blockquote></td>
+                 </tr>
+                 <tr>
+                   <td colspan="2" class="info-18" style="text-indent:50px;">F-0 microM: F + DMSO</blockquote></td>
+                 </tr>  <td colspan="2"><td>
+                 <tr>
+                   <td colspan="2" class="info-18">5.	Incubate the samples at 37°C for 4 h. </p></td>
+                 </tr>
+                 <tr>
+                   <td colspan="2" class="info-18">6.	Start preparing the flow cytometer 1 h before the end of incubation.</p>                   </td>
+                 </tr>
+                 <tr>
+                   <td colspan="2" class="info-18">7.	Take 200 microL of the sample, and centrifuge at 9000x g, 1 min., 4°C.</p>                   </td>
+                 </tr>
+                 <tr>
+                   <td colspan="2" class="info-18">8.	Remove the supernatant by using P1000 pipette.</p>                   </td>
+                 </tr>
+                 <tr>
+                   <td colspan="2" class="info-18">9.	Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend.</p>                   </td>
+                 </tr>
+                 <tr>
+                   <td colspan="2" class="info-18">10.Dispense all of each suspension into a disposable tube through a cell strainer. </p>                   </td>
+                 </tr>
+                 <tr>
+                   <td colspan="2" class="info-18">11.Measure fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).</p>                   </td>
+                 </tr>
+                 <tr>
+                   <td colspan="2">&nbsp;</td>
+                 </tr>
+                 <tr>
+                   <td colspan="2" class="entry-long">&nbsp;<a name="5" id="5"></a></td>
+                 </tr>
+                 <tr>
+                   <td colspan="2">&nbsp;</td>
+                 </tr>
+                 <tr>
+                   <td colspan="2"><h2>5. Reference</h2></td>
+                 </tr>
+                 <tr>
+                   <td colspan="2"><p class="info-18">John S. Chuang et al. (2009) Simpson’s Paradox in a Synthetic Microbial System. SCIENCE 323: 272-275</p>
                 </table>
                </div>