Difference between revisions of "Team:Pasteur Paris/Microbiology week14"

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<body>
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  <div>
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 +
  <a href="https://2016.igem.org/Team:Pasteur_Paris/Microbiology"><h2><B>Microbiology Notebook</B></h2><a/>
 +
</div>
 +
 +
<div id="home">
 +
<center></a><a href="https://2016.igem.org/Team:Pasteur_Paris/Microbiology"><img src="https://static.igem.org/mediawiki/2016/5/5a/Labwork_pasteur.png" width="40%" alt=""/></img></a></center>
 +
</div>
 +
 +
 +
<div id="week14">
 +
  <p><h5><B>Week 14</B></h5></p>
 +
 +
    <p><h3><B>September 6, 2016:</B></h3></p>
 +
    <p>
 +
        <a href="#exp1"><h4> 236. Growth of bacteria </h4></a></br>
 +
  </p>
 +
    <p><h3><B>September 7, 2016:</B></h3></p>
 +
    <p>
 +
        <a href="#exp2"><h4> 237. Purification of the protein </h4></a></br>
 +
    </p>
 +
    <p><h3><B>September 8, 2016:</B></h3></p>
 +
    <p>
 +
          <a href="#exp3"><h4> 238. Protein gel on SDS-PAGE </h4></a></br>
 +
          <a href="#exp4"><h4> 239. Harvest the culture with Miniprep </h4></a></br>
 +
 +
    </p>
 +
<p><h3><B>September 9, 2016:</B></h3></p>
 +
    <p>
 +
          <a href="#exp5"><h4> 240. Extraction of plasmid DNA </h4></a></br>
 +
          <a href="#exp6"><h4> 241. Measure the amount of DNA extracted from the miniprep </h4></a></br>
 +
 +
    </p>
 +
 +
 +
 +
    <div class="lightbox" id="exp1">
 +
      <figure>
 +
          <a href="#" class="closemsg"></a>
 +
            <figcaption> 
 +
              <p>
 +
              <U> Aim:</U> Produce our protein in BL21(DE3) competent cells, the production is induced with IPTG once it reaches an OD<sub>600 nm</sub> of 0.7.</br> </br>
 +
<U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/a/a4/T--Pasteur_Paris--Protein_induction_protocol.pdf">link</a><br/><br/>
 +
              <U>What we did in the lab:</U></br>
 +
              <U>Materials:</U></br>
 +
                  &bull; Two 2 l erlenmeyers</br>
 +
                  &bull; LB (Luria broth)</br>
 +
                  &bull; IPTG (0.1 M)</br>
 +
                  &bull; Precultures in 25 ml erlenmeyers</br>
 +
                  &bull; UV spectrophotometer (Ultrospec 3100)</br>
 +
                  &bull; Shaking incubator (INFORS HT)</br>
 +
                  &bull; Centrifuge</br>
 +
                  &bull; Buffer A (Tris-Cl 50 mM pH 7.4, NaCl 150 mM)</br> </br>
 +
 +
                  <U>Method:</U></br>
 +
                  1. Put 1 l of LB in each erlenmeyer and make them warm with the shaking incubator at 37°C and 150 rpm.<br/>
 +
                  2. Once warmed, add 12.5 ml of preculture in each erlenmeyer.<br/>
 +
                  3. Let grow in the shaking incubator.<br/>
 +
                  4. Measure the absorbance with the UV spectrophotometer every 30 minutes.</br> </br>
 +
 +
                <table>
 +
                  <caption align="bottom" align="center"><i><p> <U>Table 141 :</U> Optical Density </p></i></caption>
 +
                    <thead>
 +
                        <tr>
 +
                            <th>Time</th>
 +
                            <th>C2 (1)</th>
 +
                            <th>C2 (2)</th>
 +
                        </tr>
 +
                  </thead>
 +
                    <tbody>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>10:30 <sub>AM</sub></p></strong></td>
 +
                            <td align="center"; valign="center">0</td>
 +
                            <td align="center"; valign="center">0 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>01:55 <sub>PM</sub></p></strong></td>
 +
                            <td align="center"; valign="center">0.438</td>
 +
                            <td align="center"; valign="center">/</td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>2:25 <sub>PM</sub></p></strong></td>
 +
                            <td align="center"; valign="center">0.602</td>
 +
                            <td align="center"; valign="center">0.429 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>2:45 <sub>PM</sub></p></strong></td>
 +
                            <td align="center"; valign="center">0.679</td>
 +
                            <td align="center"; valign="center">0.600 </td>
 +
                          </tr>       
 +
                      </tbody>
 +
                  </table><br/>
 +
 +
                5. Add IPTG to reach a concentration of 0.1 mM. (3:00 <sub>PM</sub>)</br>
 +
                6. The last measure before induction is pelleted 3 min at 8000g and stored at -20°C.</br> 

 +
                7. Let induce overnight in the shaking incubator at 15°C at 150 rpm</br>
 +
                8. The day after, measure the OD<sub>600 nm</sub> and store the measure pelleted at -20°C.</br>
 +
                9. Centrifuge at 4500RPM the culture and throw out the supernatant, the pellet resuspended in 5ml of buffer A are stored at -80°C before protein extraction.</br>
 +
<br/><br/><br/>
 +
 +
            </p>
 +
          </figcaption>
 +
      </figure>
 +
    </div>
 +
      </p>
 +
 +
 +
    <div class="lightbox" id="exp2">
 +
      <figure>
 +
          <a href="#" class="closemsg"></a>
 +
            <figcaption> 
 +
              <p>
 +
              <U> Aim:</U> Purifying the protein C2 produced by BL21(DE3) using a Fast Purification Liquid Chromatography. We use the culture induced the previous day. </br> </br>
 +
<U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/0/07/T--Pasteur_Paris--FPLC_Protein_purification_protocol.pdf">link</a><br/><br/>
 +
              <U>What we did in the lab:</U></br>
 +
              <U>Materials:</U></br>
 +
                &bull; Fast Purification Liquid Chromatography </br>
 +
                &bull; Chaotropic reagent (Guanidinium HCL 6 M) </br>
 +
                &bull; EDTA 0,1 M </br>
 +
                &bull; PMSF (100 mM) </br>
 +
                &bull; Ni<sup>2+</sup> solution (100 mM) </br>
 +
                &bull; Centrifuge </br>
 +
                &bull; Buffer A (Tris 50 mM pH 7.4, NaCl 150 mM) </br>
 +
                &bull; Buffer B (Tris 50 mM pH 7.4, NaCl 150 mM, Imidazole 250 mM) </br> </br>
 +
 +
                <U>Method:</U></br>
 +
                1. Melt the pellet of bacteria C2 (from 1 l culture) and resuspend it with 10 ml of buffer A. We add 25 ml of pellet we complete until 40 ml with buffer A. </br>
 +
                2. Add 40 &#181;l of PMSF to avoid protein denaturation. </br>
 +
                3. Put the column on the FPLC, clean the FPLC with water and then fill it with buffer A. </br>
 +
                4. Sonicate the sample three times one minute at 60%, wait 90 seconds between each sonication. </br>
 +
                5. Centrifuge 25 min at 16000g (rotor Beckman JA 25.50) </br>
 +
                6. Inject your sample in the FPLC </br>
 +
                7. Get back several samples: </br>
 +
                  &emsp; &#8594; C: Crude extract : before centrigugation </br>
 +
                  &emsp; &#8594; P: Pellet </br>
 +
                  &emsp; &#8594; SN: Supernatant </br>
 +
                  &emsp; &#8594; F: Flow through (unfixed proteins) </br>
 +
                  &emsp; &#8594; W: Wash 5% of buffer B </br>
 +
                  &emsp; &#8594; Fractions (depending on the gradient) </br>
 +
<br/><br/><br/>
 +
              </p>
 +
          </figcaption>
 +
      </figure>
 +
    </div>
 +
 +
 +
 +
 +
    <div class="lightbox" id="exp3">
 +
      <figure>
 +
          <a href="#" class="closemsg"></a>
 +
            <figcaption> 
 +
              <p>
 +
              <U> Aim:</U> Get the size of the protein purified thanks to FPLC in order to know if it is our protein. <br/><br/>
 +
 +
              <U>Material: </U>
 +
              &bull; SDS-PAGE chamber <br/>
 +
              &bull; SDS-PAGE gel 4-15% gradient (BIORAD) <br/>
 +
              &bull; Protein migration buffer TGS 20X <br/>
 +
              &bull; Protein ladder PAGE Ruler (Thermofisher)<br/>
 +
              &bull; Laemmli 2X <br/>
 +
              &bull; Coomassie Blue <br/>
 +
              &bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br/><br/>
 +
 +
              <U>Method:</U><br/>
 +
            1. In nine 1.5 ml Eppendorf, put 20 &#181;l of a sample and 20 &#181;l of Laemmli 2X. <br/>
 +
            2. Let denaturate the proteins 5 minutes at 95°C. <br/>
 +
              3. Place the gel into the chamber and fill it with migration buffer. <br/>
 +
              4. Follow the next deposit table: <br/><br/>
 +
 +
              &emsp; Fraction 13 &#124; Fraction 15 &#124; Fraction 17 &#124; Fraction 19 &#124; Fraction 21 &#124; Fraction 23 &#124; Fraction 25 &#124; Fraction 27 &#124; Protein gene ruler (8 &#181;l)</br><br/>
 +
 +
              5. Launch the migration at 130V. <br/>
 +
              6. Wash the gel three times with distilled water during 5 minutes. <br/>
 +
              7. Color the gel with Coomassie Blue diluted 1/5 during 30 minutes. <br/>
 +
              8. Wash with distilled water for 5 minutes then let wash 15 minutes. <br/>
 +
              <br/><br/><br/>
 +
              </p>
 +
          </figcaption>
 +
      </figure>
 +
    </div>
 +
 +
 +
 +
 +
    <div class="lightbox" id="exp4">
 +
      <figure>
 +
          <a href="#" class="closemsg"></a>
 +
            <figcaption> 
 +
              <p>
 +
              <U> Aim:</U> To start a culture for Miniprep of insert A1 / A2 / B1 / B2 / C1 / C2 / D1 / D2 / E1 / E2. <br/>
 +
                In order to obtain a large amount of plasmid, we need to grow the bacteria overnight.<br/><br/>
 +
              <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/4/4b/T--Pasteur_Paris--Bacterial_culture_protocol.pdf">link</a><br/><br/>
 +
              <U>What we did in the lab:</U><br/>
 +
              <U>Materials:</U><br/>
 +
                &bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br/>
 +
                &bull; 15 ml Falcons <br/>
 +
                &bull; Carbenicillin 50 mg/ml <br/>
 +
                &bull; LB medium <br/><br/>
 +
 +
                <U>Method:</U><br/>
 +
                1. One colony is picked from the plates and shaken in 5.0 ml of LB supplemented with Carbenicillin at 50 &#181;g/ml. <br/>
 +
                2. The flask is placed in a shaking incubator at 37°C and 150 rpm overnight. <br/>
 +
<br/><br/><br/>
 +
              </p>
 +
            </figcaption>
 +
      </figure>
 +
    </div>
 +
 +
 +
    <div class="lightbox" id="exp5">
 +
      <figure>
 +
          <a href="#" class="closemsg"></a>
 +
            <figcaption> 
 +
              <p>
 +
              <U> Aim:</U> To perform a Midiprep to isolate plasmid DNA of pET43.1(a+) with the inserts A1 / A2 / B1 / B2 / C1 / C2 / D1 / D2 / E1 / E2 from cultures made on the 8/09. <br/>
 +
The amplification method to increase the amount of plasmid is called Midiprep. <br/>
 +
<U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/d/d5/T--Pasteur_Paris--Miniprep_protocol.pdf">link</a><br/><br/>
 +
              <U>What we did in the lab:</U><br/>
 +
              <U>Materials:</U><br/>
 +
              &bull; 50 ml Falcon tube <br/>
 +
              &bull; Shaking incubator (INFORS HT) <br/>
 +
              &bull; Swing bucket centrifuge (JOUAN GR41) <br/>
 +
              &bull; QIAGEN Miniprep kit <br/>
 +
              &bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br/><br/>
 +
 +
            <U>Method:</U><br/>
 +
              1. The protocol in step 1 ask for spinning at 6000g but we can only achieve 3500g so we used 3500g for 8 minutes. We will follow most of the protocol of QIAGEN Miniprep 2016 except for a few modifications, which we describe, therefore, below. <br/>
 +
          2. Follow QIAGEN kit steps. Final volume: 100 &#181;l <br/>
 +
<br/><br/><br/>
 +
            </p>
 +
          </figcaption>
 +
      </figure>
 +
    </div>
 +
 +
 +
    <div class="lightbox" id="exp6">
 +
      <figure>
 +
          <a href="#" class="closemsg"></a>
 +
            <figcaption> 
 +
              <p>
 +
              <U> Aim:</U> Measure the quantity of plasmid using a Nanodrop (Thermofisher) <br/><br/>
 +
 +
              <U>What we did in the lab:</U><br/>
 +
              <U>Materials:</U><br/>
 +
                    &bull; Nanodrop (Thermofisher) <br/>
 +
                    &bull; Elution buffer from QIAGEN kit <br/>
 +
                    &bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>)<br/><br/>
 +
 +
                <U>Method:</U><br/>
 +
                1. Analyze absorbance at 260nm. <br/>
 +
                2. Clean the Nanodrop with water. <br/>
 +
                3. Make the blank with 1 &#181;l of elution buffer. <br/>
 +
                4. Put 1 &#181;l of your sample on the Nanodrop. <br/>
 +
                5. Make the measure and clean the Nanodrop between each measure. <br/><br/>
 +
 +
                <U>Results:</U><br/>
 +
                <table>
 +
<caption align="bottom" align="center"><i><p><U>Table 142 :</U> Absorbance</p></i></caption>
 +
                    <thead>
 +
                        <tr>
 +
                            <th>Absorbance at 260nm</th>
 +
                            <th>A<sub>260</sub></th>
 +
                            <th>A<sub>280</sub></th>
 +
                            <th>A<sub>260/280</sub></th>
 +
                            <th>Concentration (ng/&#181;l)</th>
 +
                        </tr>
 +
                  </thead>
 +
                    <tbody>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>A1</p></strong></td>
 +
                            <td align="center"; valign="center">0.645</td>
 +
                            <td align="center"; valign="center">0.345</td>
 +
                            <td align="center"; valign="center">1.87</td>
 +
                            <td align="center"; valign="center">32.3</td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>A1’ <sub>(diluted 1/2)</sub></p></strong></td>
 +
                            <td align="center"; valign="center">1.314</td>
 +
                            <td align="center"; valign="center">0.704</td>
 +
                            <td align="center"; valign="center">1.86 </td>
 +
                            <td align="center"; valign="center">65.7 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>A2<sub>(diluted 1/4)</sub></p></strong></td>
 +
                            <td align="center"; valign="center">1.404</td>
 +
                            <td align="center"; valign="center">0.752</td>
 +
                            <td align="center"; valign="center">1.87 </td>
 +
                            <td align="center"; valign="center">70.2</td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>A2’<sub>(diluted 1/4)</sub></p></strong></td>
 +
                            <td align="center"; valign="center">0.494</td>
 +
                            <td align="center"; valign="center">0.269</td>
 +
                            <td align="center"; valign="center">1.87</td>
 +
                            <td align="center"; valign="center">24.7</td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>C1<sub>(diluted 1/4)</sub></p></strong></td>
 +
                            <td align="center"; valign="center">0.305 </td>
 +
                            <td align="center"; valign="center">0.165</td>
 +
                            <td align="center"; valign="center">1.85 </td>
 +
                            <td align="center"; valign="center">15.2 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>C1’<sub>(diluted 1/4)</sub></p></strong></td>
 +
                            <td align="center"; valign="center">0.812 </td>
 +
                            <td align="center"; valign="center">0.445</td>
 +
                            <td align="center"; valign="center">1.83</td>
 +
                            <td align="center"; valign="center">40.6</td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>C2<sub>(diluted 1/4)</sub></p></strong></td>
 +
                            <td align="center"; valign="center">0.499 </td>
 +
                            <td align="center"; valign="center">0.272</td>
 +
                            <td align="center"; valign="center">1.83</td>
 +
                            <td align="center"; valign="center">24.9 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>C2’<sub>(diluted 1/4)</sub></p></strong></td>
 +
                            <td align="center"; valign="center">0.757 </td>
 +
                            <td align="center"; valign="center">0.402</td>
 +
                            <td align="center"; valign="center">1.88</td>
 +
                            <td align="center"; valign="center">37.8</td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>D1<sub>(diluted 1/4)</sub></p></strong></td>
 +
                            <td align="center"; valign="center">0.441 </td>
 +
                            <td align="center"; valign="center">0.233</td>
 +
                            <td align="center"; valign="center">1.89</td>
 +
                            <td align="center"; valign="center">22.1 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>D1’<sub>(diluted 1/4)</sub>)</p></strong></td>
 +
                            <td align="center"; valign="center">0.725</td>
 +
                            <td align="center"; valign="center">0.396</td>
 +
                            <td align="center"; valign="center">1.83</td>
 +
                            <td align="center"; valign="center">36.3</td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>D2<sub>(diluted 1/4)</sub>)</p></strong></td>
 +
                            <td align="center"; valign="center">0.264 </td>
 +
                            <td align="center"; valign="center">0.141</td>
 +
                            <td align="center"; valign="center">1.87</td>
 +
                            <td align="center"; valign="center">13.2</td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>D2’<sub>(diluted 1/4)</sub>)</p></strong></td>
 +
                            <td align="center"; valign="center">0.418 </td>
 +
                            <td align="center"; valign="center">0.216</td>
 +
                            <td align="center"; valign="center">1.94</td>
 +
                            <td align="center"; valign="center">20.9 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>E1<sub>(diluted 1/4)</sub>)</p></strong></td>
 +
                            <td align="center"; valign="center">0.487 </td>
 +
                            <td align="center"; valign="center">0.266</td>
 +
                            <td align="center"; valign="center">1.83</td>
 +
                            <td align="center"; valign="center">24.3</td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>E1’<sub>(diluted 1/4)</sub>)</p></strong></td>
 +
                            <td align="center"; valign="center">0.478 </td>
 +
                            <td align="center"; valign="center">0.247</td>
 +
                            <td align="center"; valign="center">1.94</td>
 +
                            <td align="center"; valign="center">24.0</td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>E2<sub>(diluted 1/4)</sub>)</p></strong></td>
 +
                            <td align="center"; valign="center">1.364</td>
 +
                            <td align="center"; valign="center">0.742</td>
 +
                            <td align="center"; valign="center">1.84</td>
 +
                            <td align="center"; valign="center">68.2</td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>E2’<sub>(diluted 1/4)</sub>)</p></strong></td>
 +
                            <td align="center"; valign="center">1.041 </td>
 +
                            <td align="center"; valign="center">0.562</td>
 +
                            <td align="center"; valign="center">1.85</td>
 +
                            <td align="center"; valign="center">52.1</td>
 +
                          </tr>
 +
                            </tbody>
 +
                  </table><br/>
 +
<br/><br/><br/>
 +
 +
            </p>
 +
          </figcaption>
 +
      </figure>
 +
    </div>
 +
 +
</body>
 +
 +
</html>

Latest revision as of 03:47, 20 October 2016