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| - | <img src="https://static.igem.org/mediawiki/2014/thumb/b/b7/Tokyo_Tech_Logo.png/613px-Tokyo_Tech_Logo.png" height="150" /></h1></div>
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| - | <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>
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| - | <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>
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| - | <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>
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| - | <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>
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| - | <h2 class="title">Experiment</h2>
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| - | <span class="meta">3oxoC12HSL-dependent C4HSL production</span>
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| - | <div class="entry-long">
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| - | <p> </p>
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| - | <table width="900" border="0">
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| - | <td width="890"><div align="center" class="title-small">3OC12HSL-dependent C4HSL production module</div></td>
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| - | <td> </td>
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| - | <td><h2>1. Summary of the experiment </h2></td>
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| - | <td><p class="info-18">Construction of the 3OC12HSL-dependent C4HSL production and chloramphenicol resistance expression module</p> </td>
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| - | <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>
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| - | <td><p class="info-18">For construction of the 3OC12HSL-dependent chloramphenicol resistance (CmR) and C4HSL production module, we constructed a new part Plux-CmR-rhlI (BBa_K1529265). Plux-CmR-RhlI cell is an engineered E.coli that contains a 3OC12HSL-dependent rhlI generator and a constitutive luxR generator. We constructed a new Biobrick part Plux-CmR-rhlI by combining Plux-CmR (BBa_K39562) and rhlI (BBa_). As a constitutive luxR generator, we used Pret-luxR (BBa_S0319). In our bank story, this part is customer.</p> </td>
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| - | <td><p class="head">1-1 3OC12HSL-dependent C4HSL production </p></td>
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| - | <td><p class="info-18">First, we performed a reporter assay by using rhl reporter cell to characterize the function of 3OC12HSL-dependent C4HSL production. As the negative control of C4HSL production, we prepared C4HSL non-producer cell. C4HSL non-producer cell contains PlacIq-CmR instead of Plux-CmR-rhlI. The cell of negative control does not produce C4HSL even in the presence of 3C12HSL.</p></td>
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| - | <td> </td>
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| - | <td><p class="info-18">Sender</p></td>
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| - | <td> </td>
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| - | <td><div align="center"><img src="http://sg.openrice.com/images/v4/previewimg/sr1-icon-noResult.png" alt="" width="600" /></div></td>
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| - | <td><div align="center"><img src="http://sg.openrice.com/images/v4/previewimg/sr1-icon-noResult.png" alt="" width="600" /></div></td>
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| - | <td><div align="center"><img src="http://sg.openrice.com/images/v4/previewimg/sr1-icon-noResult.png" alt="" width="600" /></div></td>
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| - | <td> </td>
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| - | <td><p class="info-18">Repoter</p></td>
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| - | <td> </td>
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| - | <td><div align="center"><img src="http://sg.openrice.com/images/v4/previewimg/sr1-icon-noResult.png" alt="" width="600" /></div></td>
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| - | <td><div align="center"><img src="http://sg.openrice.com/images/v4/previewimg/sr1-icon-noResult.png" alt="" width="600" /></div></td>
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| - | <td><div align="center"><img src="http://sg.openrice.com/images/v4/previewimg/sr1-icon-noResult.png" alt="" width="600" /></div></td>
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| - | <td> </td>
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| - | <td><p class="info-18">We prepared four culture conditions as follow.</p></td>
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| - | <td class="info-18"> A) Culture containing Plux-CmR-RhlI cell with 3OC12HSL induction</td>
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| - | <td class="info-18"> B) Culture containing Plux-CmR-RhlI cell without 3OC12HSL induction</td>
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| - | <td class="info-18"> C) Culture containing Plux-CmR cell with 3OC12HSL induction</td>
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| - | </tr>
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| - | <td class="info-18"> D) Culture containing Plux-CmR cell without 3OC12HSL induction</td>
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| - | <td><p class="info-18">The supernatants of this four different culture were used as the inducer in the reporter assay. </p></td>
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| - | <td> </td>
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| - | <td><p class="info-18">In the reporter assay, we used a Rhl reporter strain that contains Ptet-rhlR 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>
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| - | <td> </td>
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| - | <td><p class="head">1-2 3OC12HSL-dependent growth</p></td>
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| - | <td><p class="info-18">The cell which contains Plux-CmR-rhlI can grow in the medium even containing chloramphenicol .( Chloramphenicol is one of the antibiotics. )
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| - | </p> </td>
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| - | </tr>
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| - | <td><p class="info-18">After induction, we added chloramphenicol into the medium and measured optical density hourly after induction. </p></td>
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| - | <td> </td>
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| - | <td><h2>2. Results</h2></td>
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| - | </tr>
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| - | <td> </td>
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| - | <td><p class="head">2-1 3OC12HSL-dependent C4HSL production</p></td>
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| - | </tr>
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| - | <td><p class="info-18">We measured GFP expression in the reporter cell by flow cytometer</p></td>
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| - | <td><p class="head">2-2 3OC12HSL-dependent growt</p> </td>
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| - | <td><p class="info-18">After induction, optical density were measured to estimate the concentration of the cell.</p></td>
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| - | <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-1-1.png"><img src="https://static.igem.org/mediawiki/2014/6/62/Tokyo_Tech_3-1-1.png" alt="" width="450" /></a></div></td>
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| - | <td><div align="center">Fig. 3-2-1 </div></td>
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| - | <td> </td>
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| - | <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-1-2.png"><img src="https://static.igem.org/mediawiki/2014/b/be/Tokyo_Tech_3-1-2.png" alt="" width="450" /></a></div></td>
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| - | <td><div align="center">Fig. 3-2-2 </div></td>
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| - | <td> </td>
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| - | <td><p class="info-18">As Fig 3-2-1 shows, the cell containing Plux-CmR-RhlI can grow with induction of 3OC12SL, but can’t without induction. Plux-CmR-RhlI cell grows more slowly than the positive control. The amount of CmR is lower than the positive control, Compared Fig 3-2-1 to Fig 3-2-2, With Cm, the cell grow more slowly than without Cm. geExpression of CmR in Plux-CmR-RhlI depende on induction by adding 2OC12HSL. </p> </td>
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| - | <td><h2>3. Materials and methods</h2></td>
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| - | <td> </td>
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| - | <td><p class="head">3-1 Construction</p></td>
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| - | <td><p class="head">-Strain</p></td>
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| - | <td><p class="info-18">All the samples were JM2.300 strain</p></td>
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| - | <td><p class="head">-Plasmids</p></td>
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| - | <td> </td>
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| - | <td><p class="head">3-2 Protocol </p></td>
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| - | <td><p class="info-18" style="text-indent:0px;">3-2-1. 3OC12HSL-dependent C4HSL production assay by using reporter assay</p></td>
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| - | <td><p class="info-18"><strong>Prepare the supernatant of the sender cell</strong></p></td>
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| - | <td class="info-18">1. Grow the colony of sender cell in LB containing antibiotic O/N at 37°C.</td>
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| - | <td class="info-18">2. Make a 1:100 dilution in 3 mL of fresh LB containing antibiotic and grow the cells at 37°C <br />
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| - | until the observed OD590 reaches 0.5.</td>
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| - | <td class="info-18">3. Centrifuge 1mL of the sample at 5000g, RT for 1 minute.</td>
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| - | <td class="info-18">4. Suspend the pellet in <u>1 mL of LB containing Ampicillin（50μg/mL）and Kanamycin (30μg/mL)</u></td>
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| - | <td class="info-18">5. Add 30µL of suspension in the following medium.</td>
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| - | <td class="info-18"> Add 3µL of 5µM C12HSL to 3mL LB containing Amp and Kan</td>
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| - | <td class="info-18"> Add 3µL DMSO to 3 µM of LB containing Amp and Kan</td>
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| - | <td class="info-18">6. Grow the samples of sender cell at 37°C for 4 hours.</td>
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| - | <td class="info-18">7. Measure optical density every hour (If optical density is over 1.0, dilute the cell medium.)</td>
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| - | <td class="info-18">8. Centrifuge sample at 9000g, 4°C for 1minute.Filter sterilize supernatant.</td>
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| - | <td class="info-18">9. Use the supernatant in reporter assay</td>
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| - | <td class="info-18"> </td>
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| - | <td><p class="info-18"><strong>Reporter Assay</strong></p></td>
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| - | </tr>
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| - | <tr>
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| - | <td class="info-18">1. Grow the colony of Reporter cell(D～F) in LB containing antibiotic O/N at 37°C.</td>
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| - | </tr>
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| - | <td class="info-18">2. Make a 1:100 dilution in 3 mL of fresh LB+ antibiotic and grow the cells at 37C until you reach an 0.5 OD590.(fresh culture)</td>
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| - | <td class="info-18">3. Centrifuge sample at 5000g, 25°C RT for 1 minute. Discard the supernatant.</td>
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| - | <td class="info-18">4. Suspended the sample in 3 mL of LB containing Ampicillin(50μg/mL) and Kanamycin(30μg/mL)</td>
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| - | <td class="info-18">5. Add 30µL of suspension in the following medium.</td>
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| - | <td class="info-18"> Filtrate of A①＋3mL of LB containing Amp and Kan</td>
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| - | <td class="info-18"> Filtrate of A②＋3mL of LB containing Amp and Kan</td>
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| - | <td class="info-18"> Filtrate of B①＋3mL of LB containing Amp and Kan</td>
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| - | <td class="info-18"> Filtrate of B②＋3mL of LB containing Amp and Kan</td>
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| - | <td class="info-18"> Filtrate of C①＋3mL of LB containing Amp and Kan</td>
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| - | <td class="info-18"> Filtrate of C②＋3mL of LB containing Amp and Kan</td>
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| - | <td class="info-18"> C4HSL＋3mL of LB containing Amp and Kan</td>
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| - | <td class="info-18"> DMSO + 3mL of LB containing Amp and Kan</td>
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| - | <td class="info-18">6. Grow the samples of Reporter cell in incubator at 37°C for 4 hours.</td>
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| - | <td class="info-18">7. Start preparing the flow cytometer 1 h before the end of incubation.</td>
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| - | <td class="info-18">8. Take 200 microL of the sample, and centrifuge at 9000 Xg, 1 min, 4°C.</td>
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| - | <tr>
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| - | <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"> </td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td class="head"><p>3-2-2. 3OC12HSL-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 30µL of suspension in the following medium.</td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td class="info-18"> Add 3µL of 5µM C12HSL to 3mL LB containing Amp,Kan(concentration is described upper) and Cm(100 microg /mL)</td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td class="info-18"> Add 3µ 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>
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| - | </tr>
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| - | <tr>
| + | |
| - | <td class="info-18"> </td>
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| - | </tr>
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| - | <tr>
| + | |
| - | <td><h2>4. Reference</h2></td>
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| - | </tr>
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| - | <tr>
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| - | <td class="info-18"> </td>
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| - | </tr>
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| - | </table>
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| - | <p> </p>
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