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

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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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<h2 class="title">Experiment</h2>
 
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                <span class="meta">3OC12HSL-dependent C4HSL production</span>
 
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    <div class="entry-long">
 
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              <p>&nbsp;</p>
 
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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>&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 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>&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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                </tr>
 
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                  <td>&nbsp;</td>
 
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                  <td><p class="info-18">Repoter</p></td>
 
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                </tr>
 
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                <tr>
 
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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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                </tr>
 
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                  <td>&nbsp;</td>
 
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                </tr>
 
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                  <td><p class="info-18">We prepared four culture conditions as follow.</p></td>
 
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                </tr>
 
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                <tr>
 
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                  <td class="info-18">    A) Culture containing  Plux-CmR-RhlI cell with 3OC12HSL induction</td>
 
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                </tr>
 
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                <tr>
 
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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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                  <td class="info-18">    D) Culture containing  Plux-CmR cell without 3OC12HSL induction</td>
 
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                </tr>
 
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                <tr>
 
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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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                </tr>
 
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                  <td>&nbsp;</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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                </tr>
 
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                <tr>
 
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                  <td>&nbsp;</td>
 
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                  <td><p class="head">1-2  3OC12HSL-dependent growth</p></td>
 
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                </tr>
 
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                <tr>
 
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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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                </tr>
 
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                <tr>
 
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                  <td>&nbsp;</td>
 
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                  <td><h2>2.  Results</h2></td>
 
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                <tr>
 
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                  <td>&nbsp;</td>
 
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                </tr>
 
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                <tr>
 
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                  <td><p class="head">2-1  3OC12HSL-dependent C4HSL production</p></td>
 
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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>&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="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>&nbsp;</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>&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 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 30&#181;L of suspension in the following medium.</td>
 
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                  <td class="info-18">           Add 3&#181;L of 5&#181;M C12HSL to 3mL LB containing Amp and Kan</td>
 
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                  <td class="info-18">           Add 3&#181;L DMSO to 3 &#181;M 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">&nbsp;</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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                  <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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                  <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 30&#181;L of suspension in the following medium.</td>
 
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                <tr>
 
-
                  <td class="info-18">           Filtrate  of A①+3mL of  LB containing Amp and Kan</td>
 
-
                </tr>
 
-
                <tr>
 
-
                  <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>
 
-
                <tr>
 
-
                  <td class="info-18">           Filtrate  of C①+3mL of  LB containing Amp and Kan</td>
 
-
                </tr>
 
-
                <tr>
 
-
                  <td class="info-18">          Filtrate  of C②+3mL of  LB containing Amp and Kan</td>
 
-
                </tr>
 
-
                <tr>
 
-
                  <td class="info-18">          C4HSL+3mL of LB containing Amp and Kan</td>
 
-
                </tr>
 
-
                <tr>
 
-
                  <td class="info-18">          DMSO +  3mL of LB containing Amp and Kan</td>
 
-
                </tr>
 
-
                <tr>
 
-
                  <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>
 
-
                <tr>
 
-
                  <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">&nbsp;</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 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 3&#181; 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>
 
-
                <tr>
 
-
                  <td class="info-18">&nbsp;</td>
 
-
                </tr>
 
-
                <tr>
 
-
                  <td><h2>4. Reference</h2></td>
 
-
                </tr>
 
-
                <tr>
 
-
                  <td class="info-18">&nbsp;</td>
 
-
                </tr>
 
-
              </table>
 
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              <p>&nbsp;</p>
 
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Revision as of 11:05, 12 October 2014