Team:Tokyo Tech/Experiment/Symbiosis confirmation by co-culture

From 2014.igem.org

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                 <span class="meta">Symbiosis confirmation by co-culture</span>
                 <span class="meta">Symbiosis confirmation by co-culture</span>
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<p>Under Construction！</p>
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<p>&nbsp;</p>
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                  <td colspan="2"><div align="center" class="title-small">Symbiosis confirmation by co-culture</div></td>
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<div align="center"><img src="http://www.actmp2014.com/images/under_construction%20(1).png" /></div>
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                  <td colspan="2"><h1>1. Summary of the experiment </h1></td>
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                  <td colspan="2"><p class="info-18">Rhl promoter (Prhl) is a  regulatory part activated by RhlR in the presence of N-butyryl-homoserine  lactone (also known as C4-HSL). Existing Rhl promoter (BBa_R0071) has a low  expression level even when it is activated. </p></td>
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                  <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 (PPrhl_RR: BBa_K1529320). </p></td>
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                  <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  showed about ???-folds higher in the fluorescence than that of the original Prhl  (BBa_R0071) (See results).</p></td>
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                  <td colspan="2"><p class="info-18">However, our Prhl_RR showed significant leak in the  absence of C4HSL(See results). 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 upper RhlR binding site of Prhl_RR to  Lux binding site (Prhl_LR) or the latter RhlR binding site to Lux binding site  (Prhl_RL).</p></td>
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                  <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 showed  higher maximum expression level, but also showed significant leak like Prhl_RR.  On the other hand, Prhl_RL had less leak while keeping the high expression  level (See results).</p></td>
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                  <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>
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                  <td colspan="2"><div align="center">Fig. 3-4-1. The design of our Rhl promoters</div></td>
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                  <td colspan="2"><h1>2. Results</h1></td>
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                  <td colspan="2"><p class="info-18">We measured 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. Each promoter was tested in the  presence and also in the absence of C4HSL (See Materials and Methods for  detailed procedures).</p></td>
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                  <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig._3-4-2.png"><img src="https://static.igem.org/mediawiki/2014/thumb/3/3d/Tokyo_Tech_Fig._3-4-2.png/588px-Tokyo_Tech_Fig._3-4-2.png" alt="" width="450" /></a></div></td>
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                  <td colspan="2"><div align="center">Fig. 3-4-2. The four promoters we tested</div></td>
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                  <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 Prhl, Prhl_RR,  and Prhl_RL</p></td>
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                  <td colspan="2"><p class="info-18">As Fig 3-4-4 shows, when  C4HSL is induced, Prhl_RR showed higher maximum expression level and higher leak  than the original Prhl.</p>                  </td>
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                  <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>
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                  <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>
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                  <td><div align="center">Fig. 3-4-3. The Fluorescence intensity of the cells  <br />
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                  (with positive and negative controls)</div></td>
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                  <td><div align="center">Fig. 3-4-4. The fluorescence intensity of the cells  with original Prhl (BBa_R0071), Prhl_RR (BBa_K1529320), Prhl_RL (BBa_K1529300)</div></td>
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                  <td colspan="2"><h1>3. Materials and methods</h1></td>
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                  <td colspan="2" class="head">3-1. Construction</td>
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                  <td colspan="2" class="info-24">-Strain</td>
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                  <td colspan="2"><p class="info-18">     All the samples were JM2.300 strain </p></td>
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                  <td colspan="2" class="info-24">-Plasmids</td>
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                  <td colspan="2"><p class="info-18">     A. Ptet-RhlR (pSB6A1), Prhl-GFP (pSB3K3)</p></td>
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                  <td colspan="2"><div align="center">
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                    <blockquote>
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                      <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="800" /></a></p>
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                  <td colspan="2"><div align="center">Fig. 3-4-5 </div></td>
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                  <td colspan="2"><p class="info-18">     B. Ptet-RhlR (pSB6A1)　Prhl_RR-GFP (pSB3K3)</p></td>
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                  <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="800" /></a></div></td>
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                  <td colspan="2"><div align="center">Fig. 3-4-6 </div></td>
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                  <td colspan="2"><p class="info-18">     C. Ptet-RhlR (pSB6A1)　Prhl_LR-GFP (pSB3K3)</p></td>
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                  <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="800" /></div></td>
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                  <td colspan="2"><div align="center">Fig. 3-4-7</div></td>
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                  <td colspan="2"><p class="info-18">     D. Ptet-RhlR (pSB6A1)　Prhl_RL-GFP (pSB3K3)</p></td>
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                  <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="800" /></a></div></td>
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                  <td colspan="2"><p class="info-18">     E. Ptet-RhlR (pSB6A1)　PlacUV5-GFP (pSB3K3) ...Positive control</p></td>
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                  <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="800" /></a></div></td>
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                  <td colspan="2"><div align="center">Fig. 3-4-9</div></td>
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                  <td colspan="2"><p class="info-18">     F. Ptet-RhlR (pSB6A1)　promoter  less-GFP(pSB3K3) ...Negative control</p></td>
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                  <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>
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                  <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>
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                  <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>
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                  <td colspan="2"><p class="info-18">4. Add 30 microL of 500 microM C4HSL or DMSO as listed below:</p>                  </td>
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                  <td colspan="2"><blockquote class="info-18">                    A-5 μM: A + C4HSL</blockquote></td>
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                  <td colspan="2"><blockquote class="info-18">                    A-0 μM: A + DMSO</blockquote></td>
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                  <td colspan="2"><blockquote class="info-18">F-0  μM: F + DMSO</blockquote></td>
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                  <td colspan="2"><p class="info-18">5. Incubate the samples at 37°C for 4 h.</p></td>
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                  <td colspan="2"><p class="info-18">6. Start preparing the flow cytometer 1 h before the end of incubation.</p></td>
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                  <td colspan="2"><p class="info-18">7. Take 200 microL of the sample, and centrifuge at 9000 Xg, 1 min,  4°C.</p></td>
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                  <td colspan="2"><p class="info-18">8. Remove the supernatant by using P1000 pipette.</p></td>
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                  <td colspan="2"><p class="info-18">9. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend.</p></td>
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                  <td colspan="2"><p class="info-18">10. Dispense all of each suspension into a disposable tube through a  cell strainer. </p></td>
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                  <td colspan="2"><p class="info-18">11. Measure fluorescence intensity with a flow cytometer (We used BD  FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).</p></td>
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                  <td colspan="2"><h1>4. References</h1></td>
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                  <td colspan="2"><p class="info-18">[1] “Simpson’s Paradox in a Synthetic  Microbial System” John S. Chuang, Olivier Rivoire, Stanislas Leibler 9 JANUARY  2009 VOL 323 SCIENCE</p></td>
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Revision as of 14:20, 7 October 2014

Tokyo_Tech

Retrieved from "http://2014.igem.org/Team:Tokyo_Tech/Experiment/Symbiosis_confirmation_by_co-culture"