Team:Tokyo Tech/Experiment/C4HSL-dependent 3oxoC12HSL production

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		<h2 class="title">Experiment</h2>
                <span class="meta">C4HSL-dependent 3OC12HSL production</span>
                <div class="entry-long">
               <p>&nbsp;</p>
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                   <td width="890"><div align="center" class="title-small">C4HSL-dependent 3OC12HSL production module</div></td>
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                   <td>&nbsp;</td>
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                   <td>&nbsp;</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 C4HSL-dependent 3OC12HSL 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 C4HSL-dependent chloramphenicol resistance (CmR) and 3OC12HSL production module, we constructed a new part Plux-CmR-lasI (BBa_). Plux-CmR-lasI cell is an engineered E.coli that contains a C4HSL-dependent lasI generator and a constitutive rhlR generator. We constructed a new Biobrick part Plux-CmR-lasI by combining Plux-CmR (BBa_K39562) and lasI (BBa_). As a constitutive rhlR generator, we used Pret-rhlR (BBa_S0319). In our bank story, this part is company.                   </p></td>
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                   <td><p class="head">1-1 C4HSL-dependent 3OC12HSL production </p></td>
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                   <td><p class="info-18">First, we performed a reporter assay by using Lux reporter cell to characterize the function of C4HSL-dependent 3OC12HSL production. As the negative control of 3OC12HSL production, we prepared 3OC12HSL non-producer cell. 3OC12HSL non-producer cell contains Plux-CmR instead of Plux-CmR-lasI. The cell of negative control does not produce 3OC12HSL even in the presence of C4HSL.</p></td>
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                   <td>&nbsp;</td>
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                   <td><p class="info-18">Sender</p></td>
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                   <td>&nbsp;</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>&nbsp;</td>
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                   <td><p class="info-18">Repoter</p></td>
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                   <td>&nbsp;</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>&nbsp;</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-LasI cell with C4HSL induction</td>
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                   <td class="info-18">    B) Culture containing Plux-CmR-LasI cell without C4HSL induction</td>
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                   <td class="info-18">    C) Culture containing Plux-CmR cell with C4HSL induction</td>
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                   <td class="info-18">    D) Culture containing Plux-CmR cell without C4HSL 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>&nbsp;</td>
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                   <td><p class="info-18">In the reporter assay, we used a Lux reporter strain that contains Ptet-luxR 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>&nbsp;</td>
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                   <td><p class="head">1-2  C4HSL-dependent growth</p></td>
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                   <td><p class="info-18">The cell which contains Plux-CmR-lasI can grow in the medium containing chloramphenicol<br />
                   (Chloramphenicol is one of the antibiotics. )                   </p></td>
                 </tr>
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                   <td><p class="info-18">After induction, we added chloramphenicol into the medium and measured optical density<br />
                   after induction.</p></td>
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                   <td>&nbsp;</td>
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                   <td><h2>2.  Results</h2></td>
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                   <td>&nbsp;</td>
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                   <td><p class="head">2-1  C4HSL-dependent 3OC12HSL production</p></td>
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                   <td><p class="info-18">We measured the expression of GFP in the reporter cell by flow cytometer.</p></td>
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                   <td><p class="head">2-2 C4HSL-dependent growth</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="http://sg.openrice.com/images/v4/previewimg/sr1-icon-noResult.png" alt="" width="600" /></a></div></td>
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                   <td><div align="center">Fig. 3-1-1 </div></td>
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                   <td>&nbsp;</td>
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                   <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_3-1-2.png"><img src="http://sg.openrice.com/images/v4/previewimg/sr1-icon-noResult.png" alt="" width="600" /></a></div></td>
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                   <td><div align="center">Fig. 3-1-2 </div></td>
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                   <td>&nbsp;</td>
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                   <td><p class="info-18">explanation?    </p>                   </td>
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                   <td>&nbsp;</td>
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                   <td><h2>3.  Materials and methods</h2></td>
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                   <td>&nbsp;</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>&nbsp;</td>
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                   <td><p class="head">3-2 </p></td>
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                   <td><p class="info-18" style="text-indent:0px;">3-2-1.  C4HSL-dependent 3OC12HSL production assay by using reporter assay</p></td>
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                   <td><p class="info-18">Prepare the supernatant of the sender cell</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>
                 </tr>
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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 37C until the observed OD590 reaches 0.5. </td>
                 </tr>
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                   <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> </u><u>1 mL of LB containing Ampicillin</u><u>（</u><u>50μg/mL</u><u>）</u><u>and Kanamycin(30μg/mL) .</u></td>
                 </tr>
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                   <td class="info-18">5. Add 30&#181;L of suspension in the following medium.</td>
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                   <td class="info-18">           Add 30&#181;L of 500&#181;M C12HSL to 3mL LB containing Amp and Kan</td>
                 </tr>
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                   <td class="info-18">           Add 30&#181;L 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>
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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>
                 </tr>
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                   <td class="info-18">8. Centrifuge sample at 9000g, 4°C for 1minute. Filter sterilize supernatant.</td>
                 </tr>
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                   <td class="info-18">9. Use  the supernatant in reporter assay.</td>
                 </tr>
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                   <td class="info-18">&nbsp;</td>
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                   <td><p class="info-18"><strong>Reporter Assay</strong></p></td>
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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>
                 </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 37°C until you reach an 0.5 OD590. (fresh  culture).</td>
                 </tr>
                 <tr>
                   <td class="info-18">3. Centrifuge  sample at 5000g, 25°C RT for 1 minute. Discard the supernatant.</td>
                 </tr>
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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>
                 </tr>
                 <tr>
                   <td class="info-18">5. Add 30&#181;L of suspension in the following medium.</td>
                 </tr>
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                   <td class="info-18">           Filtrate  of A①＋3mL of  LB containing Amp and Kan</td>
                 </tr>
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                   <td class="info-18">           Filtrate  of A②＋3mL of  LB containing Amp and Kan</td>
                 </tr>
                 <tr>
                   <td class="info-18">           Filtrate  of B①＋3mL of  LB containing Amp and Kan</td>
                 </tr>
                 <tr>
                   <td class="info-18">           Filtrate  of B②＋3mL of  LB containing Amp and Kan</td>
                 </tr>
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                   <td class="info-18">           Filtrate  of C①＋3mL of  LB containing Amp and Kan</td>
                 </tr>
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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">          3OC12HSL＋3mL of LB containing Amp and Kan</td>
                 </tr>
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                   <td class="info-18">          DMSO +  3mL of LB containing Amp and Kan</td>
                 </tr>
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                   <td class="info-18">6. Grow  the samples of Reporter cell in incubator at 37°C for 4 hours.</td>
                 </tr>
                 <tr>
                   <td class="info-18">7. Start  preparing the flow cytometer 1 h before the end of incubation.</td>
                 </tr>
                 <tr>
                   <td class="info-18">8. Take  200 microL of the sample, and centrifuge at 9000 Xg, 1 min, 4°C.</td>
                 </tr>
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                   <td class="info-18">9. Remove  the supernatant by using P1000 pipette.</td>
                 </tr>
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                   <td class="info-18">10. Add 1 mL  of filtered PBS (phosphate-buffered saline) and suspend.</td>
                 </tr>
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                   <td class="info-18">11. Dispense  all of each suspension into a disposable tube through a cell strainer.</td>
                 </tr>
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                   <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>
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                   <td class="info-18">&nbsp;</td>
                 </tr>
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                   <td class="head"><p>3-2-2. C4HSL-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 30&#181;L of suspension in the following medium.</td>
                 </tr>
                 <tr>
                   <td class="info-18">           Add 3&#181;L of 5&#181;M C12HSL to 3mL LB containing Amp, Kan(concentration  is described upper) and Cm(100 microg /mL).</td>
                 </tr>
                 <tr>
                   <td class="info-18">           Add 3microL 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>
                 </tr>
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                   <td class="info-18">&nbsp;</td>
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                   <td><h2>4. Reference</h2></td>
                 </tr>
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                   <td class="info-18">&nbsp;</td>
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