Difference between revisions of "Team:Pasteur Paris/Microbiology week5"
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<p><h3><B>July 4, 2016:</B></h3></p> | <p><h3><B>July 4, 2016:</B></h3></p> | ||
<p> | <p> | ||
| − | <a href="#exp1"><h4> 47. | + | <a href="#exp1"><h4> 47.Digestion of pET43.1 (a) with XbaI and HindIII </h4></a></br> |
<a href="#exp2"><h4> 48. DNA measurements</h4></a></br> | <a href="#exp2"><h4> 48. DNA measurements</h4></a></br> | ||
<a href="#exp3"><h4> 49. Agarose gel (0.7 % agarose gel )</h4></a></br> | <a href="#exp3"><h4> 49. Agarose gel (0.7 % agarose gel )</h4></a></br> | ||
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</table> | </table> | ||
<center>Table 39</center> | <center>Table 39</center> | ||
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</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
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<div class="lightbox" id="exp7"> | <div class="lightbox" id="exp7"> | ||
<figure> | <figure> | ||
| − | <a href="# exp7" class="closemsg"></a> | + | <a href="#exp7" class="closemsg"></a> |
| + | <figcaption> | ||
| + | <p> | ||
| + | <U> Aim:</U>To save time, we do another dephosphorylation of pET43.1 digested by enzymes to ligate it to the insert C2 (digest by XbaI/HindIII on June,28 2016). </br></br> | ||
| + | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
| + | <U>What we did in the lab:</U></br> | ||
| + | <U>Materials:</U></br> | ||
| + | • pET43.1a plasmid digested by XbaI/HindIII</br> | ||
| + | • Dephosphorylation rSAP enzyme</br> | ||
| + | • P10 and P200 pipet, 1.5 eppendorf, water bath (37 °C and 65 °C)</br> | ||
| + | |||
| + | |||
| + | <U>Method:</U></br> | ||
| + | 1. For volumes, refer on the next table: | ||
| + | 2. We dephosphorylate 7.5*120 ng/µl, so 900 ng of pET43.1</br></br> | ||
| + | |||
| + | <table> | ||
| + | <thead> | ||
| + | <tr> | ||
| + | <th>DNA of pET43.1a(+)( 120 ng/µl)</th> | ||
| + | <th>rSAP ( µl)</th> | ||
| + | <th>Buffer 10X ( µl)</th> | ||
| + | <th>H2O ( µl)</th> | ||
| + | <th>TOTA ( µl)</th> | ||
| + | |||
| + | </tr> | ||
| + | </thead> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td>7.5</td> | ||
| + | <td>2.58</td> | ||
| + | <td>6</td> | ||
| + | <td>43.92</td> | ||
| + | <td>60</td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| + | <center>Table 39</center> | ||
| + | </p> | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </div> | ||
| + | |||
| + | <div class="lightbox" id="exp8"> | ||
| + | <figure> | ||
| + | <a href="#exp8" class="closemsg"></a> | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
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<U>Results:</U></br></br> | <U>Results:</U></br></br> | ||
</p> | </p> | ||
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<div class="lightbox" id="exp9"> | <div class="lightbox" id="exp9"> | ||
<figure> | <figure> | ||
| − | <a href="# exp9" class="closemsg"></a> | + | <a href="#exp9" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U></br></br> | + | <U> Aim:</U>At this stage, we want to obtain a recombinant vector (pET43.1a(+) + C2)</br></br> |
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
<U>Materials:</U></br> | <U>Materials:</U></br> | ||
| + | • pET43.1a(+) plasmid digested by XbaI/HindIII and dephosphorylate (done previously)</br> | ||
| + | • C2 insert cut by XbaI/HindIII (done on the june,28 2016)</br> | ||
| + | • T4 Ligase and Buffer 10X</br> | ||
| + | • P10 and P200 pipet, 1.5 eppendorf, warm bath (37 °C and 65 °C)</br> | ||
| + | |||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | < | + | 1. For volums, refer to the next table :></br>></br> |
| + | |||
| + | C2 = 11.4 ng/µl></br> | ||
| + | pET43.1 = 50 ng/µl (900 ng 60 µl)></br> | ||
| + | |||
| + | <table> | ||
| + | <thead> | ||
| + | <tr> | ||
| + | <th></th> | ||
| + | <th>1:1</th> | ||
| + | <th>1:4</th> | ||
| + | <th>Only pET43.1a(+)</th> | ||
| + | </tr> | ||
| + | </thead> | ||
| + | |||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td><strong><p>pET43.1a(+) (50mg) (µl)</p></strong></td> | ||
| + | <td>20</td> | ||
| + | <td>20</td> | ||
| + | <td>20</td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td><strong><p>Insert C2 (µl)</p></strong></td> | ||
| + | <td>1.4</td> | ||
| + | <td>4.2</td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td><strong><p>T4 ligase (µl) </p></strong></td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>Buffer 10X (µl) </p></strong></td> | ||
| + | <td>2.5</td> | ||
| + | <td>2.5</td> | ||
| + | <td>2.5</td> | ||
| + | </tr> | ||
| + | <tr> <tr> | ||
| + | <td><strong><p>H20 (µl) </p></strong></td> | ||
| + | <td>0</td> | ||
| + | <td>0</td> | ||
| + | <td>0</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>TOTAL (µl) </p></strong></td> | ||
| + | <td>24.9</td> | ||
| + | <td>27.7</td> | ||
| + | <td>22.5</td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | |||
| + | </table> | ||
| + | <center> Table 42</center></br></br></br> | ||
| + | |||
| + | Incubate 10 min at room temperature, and put it at 37 °C during 10 min. To keep it and use it after, we put it at -20 °C. </br> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
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<div class="lightbox" id="exp10"> | <div class="lightbox" id="exp10"> | ||
<figure> | <figure> | ||
| − | <a href="# exp10" class="closemsg"></a> | + | <a href="#exp10" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U></br></br> | + | <U> Aim:</U>Check if ligation of pET43.1+C1 and pET43.1+C2 were successfully done |
| + | </br></br> | ||
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
<U>Materials:</U></br> | <U>Materials:</U></br> | ||
| + | • pET43.1a plamid </br> | ||
| + | • pET43.1a plamid cutted by HindIII/XbaI</br> | ||
| + | • C1 and C2 cutted by HindIII/XbaI</br> | ||
| + | • agarose gel 0.7%</br> | ||
| + | • TAE 0.5x buffer</br> | ||
| + | • Electrophoresis generator at 130 V</br> | ||
| + | • DNA ladder (Thermoscientific gene ruler 1 kb)</br> | ||
| + | • Electrophoresis generator (at 50 V and after at 90 V)</br> | ||
| + | |||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| + | 1. Prepare an agarose gel at 0.7 % (Refer on the detail protocol which is on the protocol parts) </br> | ||
| + | 2. Fill electrophoresis chamber with TAE 0.5X</br> | ||
| + | 3. Follow the deposit table :</br></br> | ||
| + | |||
| + | <table> | ||
| + | <thead> | ||
| + | <tr> | ||
| + | <th>Lanes</th> | ||
| + | <th>L1</th> | ||
| + | <th>L2</th> | ||
| + | <th>L3</th> | ||
| + | <th>L4</th> | ||
| + | <th>L5</th> | ||
| + | <th>L6</th> | ||
| + | <th>L7</th> | ||
| + | <th>L8</th> | ||
| + | <th>L9</th> | ||
| + | </tr> | ||
| + | </thead> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td><strong><p>Name</p></strong></td> | ||
| + | <td>Marker weight</td> | ||
| + | <td>pET43.1a(+) uncul</td> | ||
| + | <td></td> | ||
| + | <td>C1 (1:1)</td> | ||
| + | <td>C1 (1:3)</td> | ||
| + | <td></td> | ||
| + | <td>C2 (1:1)</td> | ||
| + | <td>C2 (1:3)</td> | ||
| + | <td>pET43.1a(+) only</td> | ||
| + | |||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>DNA (µl)</p></strong></td> | ||
| + | <td>7</td> | ||
| + | <td>6</td> | ||
| + | <td></td> | ||
| + | <td>5</td> | ||
| + | <td>5</td> | ||
| + | <td></td> | ||
| + | <td>5</td> | ||
| + | <td>5</td> | ||
| + | <td>5</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>DNA (µl)</p></strong></td> | ||
| + | <td>0</td> | ||
| + | <td>0</td> | ||
| + | <td></td> | ||
| + | <td>0</td> | ||
| + | <td>0</td> | ||
| + | <td></td> | ||
| + | <td>0</td> | ||
| + | <td>0</td> | ||
| + | <td>0</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>DNA (µl)</p></strong></td> | ||
| + | <td>0</td> | ||
| + | <td>0</td> | ||
| + | <td></td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | <td></td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| + | <center>Table 43</center> | ||
| + | </br></br></br> | ||
| + | Let 1h at 50V and put it at 100 V.</br></br> | ||
| + | |||
<U>Results:</U></br></br> | <U>Results:</U></br></br> | ||
| + | The electrophoresis shows us that DNA wasn’t ligated, so we added 1 µl more of T4 ligase and 1 µl of buffer in each tube and we let ligate during 1h. </br></br> | ||
| + | |||
| + | After one hour, we did another electrophoresis.</br></br> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
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<div class="lightbox" id="exp11"> | <div class="lightbox" id="exp11"> | ||
<figure> | <figure> | ||
| − | <a href="# exp11" class="closemsg"></a> | + | <a href="#exp11" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U></br></br> | + | <U> Aim:</U>Check if ligation of pET43.1+C1 and pET43.1+C2 was successfully done.</br></br> |
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
<U>Materials:</U></br> | <U>Materials:</U></br> | ||
| + | • pET43.1a plamid </br> | ||
| + | • pET43.1a plamid cutted by HindIII/XbaI</br> | ||
| + | • C1 and C2 cut by HindIII/XbaI</br> | ||
| + | • agarose gel 0.7%</br> | ||
| + | • TAE 0.5x buffer</br> | ||
| + | • Electrophoresis generator at 130 V</br> | ||
| + | • DNA ladder (Thermoscientific gene ruler 1 kb)</br> | ||
| + | • P10 pipet, P20 pipet, test tube 250mL, electrophoresis BIORAD Mini-Sub Cell GT, 2 type of tips, 1.5ml Eppendorf sterile tubes, 37°C water bath, shaking incubator centrifuge 5415D, </br></br> | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| + | 1. -Fill the electrophoresis chamber with TAE 0.5X buffer</br> | ||
| + | 2. Following this deposit table :</br></br> | ||
| + | <table> | ||
| + | <thead> | ||
| + | <tr> | ||
| + | <th>Lanes</th> | ||
| + | <th>L1</th> | ||
| + | <th>L2</th> | ||
| + | <th>L3</th> | ||
| + | <th>L4</th> | ||
| + | <th>L5</th> | ||
| + | <th>L6</th> | ||
| + | <th>L7</th> | ||
| + | <th>L8</th> | ||
| + | <th>L9</th> | ||
| + | </tr> | ||
| + | </thead> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td><strong><p>Name</p></strong></td> | ||
| + | <td>Marker weight</td> | ||
| + | <td></td> | ||
| + | <td>pET43.1a(+) uncul</td> | ||
| + | <td>pET43.1a(+) only</td> | ||
| + | <td>C1 (1:1)</td> | ||
| + | <td>C1 (1:3)</td> | ||
| + | <td>C2 (1:1)</td> | ||
| + | <td>C2 (1:3)</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>DNA (µl)</p></strong></td> | ||
| + | <td>5</td> | ||
| + | <td></td> | ||
| + | <td>1</td> | ||
| + | <td>5</td> | ||
| + | <td>5</td> | ||
| + | <td>5</td> | ||
| + | <td>5</td> | ||
| + | <td>5</td> | ||
| + | <td>5</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>DNA (µl)</p></strong></td> | ||
| + | <td>0</td> | ||
| + | <td></td> | ||
| + | <td>0</td> | ||
| + | <td>0</td> | ||
| + | <td>0</td> | ||
| + | <td>0</td> | ||
| + | <td>0</td> | ||
| + | <td>0</td> | ||
| + | <td>0</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>DNA (µl)</p></strong></td> | ||
| + | <td>0</td> | ||
| + | <td></td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| + | <center>Table 44</center> | ||
<U>Results:</U></br></br> | <U>Results:</U></br></br> | ||
</p> | </p> | ||
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</figure> | </figure> | ||
</div> | </div> | ||
| − | |||
<div class="lightbox" id="exp12"> | <div class="lightbox" id="exp12"> | ||
<figure> | <figure> | ||
| − | <a href="# exp12" class="closemsg"></a> | + | <a href="#exp12" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> | + | <U>Results:</U></br> |
| − | + | MpET43.1 = 5 300 bp * 660 g.mol-1.bp-1</br> | |
| − | + | MpET43.1 = 3.5*10-6 g/mol</br> | |
| − | + | After condensation, we lost 5 299 molecules of water (one between each bp), so we have 0.1*106 g/mol</br></br> | |
| − | + | Finally, we have :</br> | |
| − | + | MpET43.1 = 3.4*106 g/mol</br> | |
| + | Minsert = 5.6*105 g/mol</br></br> | ||
| + | According to the instruction:</br> | ||
| + | - We have 0.12 µmol/L in 20 µl, and we must have 10 pg to have a good yield in colonies. </br> | ||
| + | - Finally, we have 0.12 * 20*10-6 = 2.4*10-6 µmol of plasmid and 8.16 pg of plasmid. </br> | ||
| + | </br> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
<div class="lightbox" id="exp13"> | <div class="lightbox" id="exp13"> | ||
<figure> | <figure> | ||
| − | <a href="# exp13" class="closemsg"></a> | + | <a href="#exp13" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U></br></br> | + | <U> Aim:</U>: To make stock cells containing the plasmids, we need to enter the plasmid with C1 and C2 insert into the bacteria DH5 competent cells.</br></br> |
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
| − | <U>Materials:</U></br> | + | <U>Materials:</U></br>competent cells, SOC media, 42°C waterbath, LB/carbenicillin 50 µg/ml.</br> |
<U>Method:</U></br> | <U>Method:</U></br> | ||
| + | 1- In five 1.5 ml eppendorf tubes, we put 40 µl of DH5 competent cells and we add 5µl of : </br> | ||
| + | (vector:Insert) ratio</br> | ||
| + | (1) (1:0) empty plasmid</br> | ||
| + | (2) C1 (1:1) </br> | ||
| + | (3) C1 (1:3) </br> | ||
| + | (4) C2 (1:1) </br> | ||
| + | (5) C2 (1:3) </br></br> | ||
| + | |||
| + | 2- Place the tubes on ice during 30 min (18h30)</br> | ||
| + | 3- Put them at 42 °C during 40 sec</br> | ||
| + | 1- Put them on ice during 2 min 30 sec</br> | ||
| + | 2- Add 200 µl of SOC in each tubes and place them in a shaking incubator for 1 h at 37 °C and at 225 RPM</br> | ||
| + | 3- We spread our mix (250 µl) on petri dishes made of LB and carbenicillin </br></br> | ||
| + | 4- Put them at 37 °C for one night</br> | ||
| + | |||
<U>Results:</U></br></br> | <U>Results:</U></br></br> | ||
</p> | </p> | ||
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</figure> | </figure> | ||
</div> | </div> | ||
| − | |||
<div class="lightbox" id="exp14"> | <div class="lightbox" id="exp14"> | ||
<figure> | <figure> | ||
| − | <a href="# exp14" class="closemsg"></a> | + | <a href="#exp14" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | + | Only two colonies have grown on the plate C2 (1:3), all the other petri dishes are empty. </br> | |
| − | + | According to the electrophoresis gel, the ligation has worked for C1 (1:3) and C2 (1:1) but it do not correspond to our results. We decided to pool C2 (1:1) with C2 (1:3) and C1 (1:1) with C1 (1:3) because the gel shows the same efficiency of ligation, in order to have more DNA for transformation. | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 769: | Line 1,041: | ||
<div class="lightbox" id="exp15"> | <div class="lightbox" id="exp15"> | ||
<figure> | <figure> | ||
| − | <a href="# exp15" class="closemsg"></a> | + | <a href="#exp15" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U></br></br> | + | <U> Aim:</U>We want to increase the number of bacteria to check if they have the right plasmid. As we have two colonies, therefore we placed them to grow in liquid media in two Falcons. </br></br> |
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
| − | |||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| + | In a 50 ml Falcon : </br> | ||
| + | - Put 10 ml of LB and 5 µl of carbenicillin at 100 mg/ml. </br> | ||
| + | - Add the colony we took with a tooth-pick. </br> | ||
| + | - Put the Falcon in a shaking incubator at 37 °C for one day. </br></br> | ||
| + | |||
<U>Results:</U></br></br> | <U>Results:</U></br></br> | ||
</p> | </p> | ||
| Line 786: | Line 1,062: | ||
<div class="lightbox" id="exp16"> | <div class="lightbox" id="exp16"> | ||
<figure> | <figure> | ||
| − | <a href="# exp16" class="closemsg"></a> | + | <a href="#exp16" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U></br></br> | + | The ligation does not work well, so we pooled our ligation products to increase the amount of available DNA. |
| + | <U> Aim:</U>: At this stage, we want to obtain a ligated vector (pET43.1a(+) + C1/C2)</br></br> | ||
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
| − | |||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | < | + | C(pET43.1) = 29.5 ng/µl</br> |
| + | C(C1) = 11.4 ng/µl</br> | ||
| + | C(C2) = 9.2 ng/µl</br></br> | ||
| + | |||
| + | Add volumes of 1µl of T4 Ligase and 1 µl of Buffer 10X in the tubes C1(1 :1)+C1(1 :3) and C2(1 :1)+C2(1 :3).</br> | ||
| + | |||
| + | Let the ligation proceed during 1h30 at RT and incubate it 10 min at 65 °C to inactivated the ligase.</br> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
| + | |||
<div class="lightbox" id="exp17"> | <div class="lightbox" id="exp17"> | ||
<figure> | <figure> | ||
| − | <a href="# exp17" class="closemsg"></a> | + | <a href="#exp17" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U></br></br> | + | <U> Aim:</U>The previuos experiment being still underway, we want to move ahead wier inserts. We want to digest our B2 insert to put it in the expression vector.</br></br> |
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
<U>Materials:</U></br> | <U>Materials:</U></br> | ||
| + | • eppendorf (0.5 ml)</br> | ||
| + | • microbiology equipement</br> | ||
| + | • B2 insert</br> | ||
| + | • Enzymes (HindIII and XbaI)</br> | ||
| + | • H2O RNAse free</br> | ||
| + | • Buffer 10X</br></br> | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | < | + | 1. Mixing all of reactants and let the digestion proceed during 1h30 at 37 °C </br> |
| + | 2. Follow this next table</br></br> | ||
| + | <table> | ||
| + | <thead> | ||
| + | <tr> | ||
| + | <th></th> | ||
| + | <th>B2</th> | ||
| + | </tr> | ||
| + | </thead> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td><strong><p>Vol DNA (50ng/µl)</p></strong></td> | ||
| + | <td>20 µl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong>Vol Hind III</strong></td> | ||
| + | <td>0.5 µl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong>A<sub>Vol XbaI</sub></strong></td> | ||
| + | <td>0.5 µl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong>Vol buffer 10X (CutSmart)</strong></td> | ||
| + | <td>2.5 µl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong>Vol TOTAL</strong></td> | ||
| + | <td>25 µl</td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| + | <center>Table 45</center></br></br> | ||
| + | |||
| + | Allow the digestion proceed for 1h and add an extra 0.5 µl of enzyme and leave the reaction going for 1h more at 37 °C.</br> | ||
| + | 1. Incubate it 10 min at 65 °C to inactivate the enzymes</br> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 818: | Line 1,142: | ||
<div class="lightbox" id="exp18"> | <div class="lightbox" id="exp18"> | ||
<figure> | <figure> | ||
| − | <a href="# exp18" class="closemsg"></a> | + | <a href="#exp18" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> | + | <U> Aim:</U>Than we do the ligation of the insert B2 with pET43.1 and the transformation of all our products of ligation B2/C1 mix and empty plasmid as control. </br></br> |
| − | + | (Refer to previous protocol)</br></br> | |
| − | + | ||
| − | + | ||
| − | + | ||
<U>Results:</U></br></br> | <U>Results:</U></br></br> | ||
</p> | </p> | ||
| Line 834: | Line 1,156: | ||
<div class="lightbox" id="exp19"> | <div class="lightbox" id="exp19"> | ||
<figure> | <figure> | ||
| − | <a href="# exp19" class="closemsg"></a> | + | <a href="#exp19" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<U>Results:</U></br></br> | <U>Results:</U></br></br> | ||
| + | B2 (1 : 1) Nothing has grown</br> | ||
| + | B2 (1 : 3) Nothing has grown</br> | ||
| + | C2 (1 : 0) Nothing has grown</br> | ||
| + | C1 mix Nothing has grown</br> | ||
| + | |||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 850: | Line 1,172: | ||
<div class="lightbox" id="exp20"> | <div class="lightbox" id="exp20"> | ||
<figure> | <figure> | ||
| − | <a href="# exp20" class="closemsg"></a> | + | <a href="#exp20" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U></br></br> | + | <U> Aim:</U>To get the plasmid back and verify that it contains inserts. |
| + | We have two colonies, so for each colony, we started two cultures in Falcon tubes. One being a primary culture and the other a backup, in case the culture doesn't grow. | ||
| + | </br></br> | ||
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
<U>Materials:</U></br> | <U>Materials:</U></br> | ||
| + | They are named :</br> | ||
| + | C2 (1 :3) 1.1</br> | ||
| + | C2 (1 :3) 1.2</br> | ||
| + | C2 (1 :3) 2.1 </br> | ||
| + | C2 (1 :3) 2.2</br></br> | ||
| + | |||
| + | NB :(The naming convention here is first number is the number of colony, and the second number correspond to primary 1 ou secondary 2 cultures).</br> | ||
| + | |||
<U>Method:</U></br> | <U>Method:</U></br> | ||
<U>Results:</U></br></br> | <U>Results:</U></br></br> | ||
| Line 866: | Line 1,198: | ||
<div class="lightbox" id="exp21"> | <div class="lightbox" id="exp21"> | ||
<figure> | <figure> | ||
| − | <a href="# exp21" class="closemsg"></a> | + | <a href="#exp21" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | |||
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
| − | |||
| − | |||
| − | |||
| − | |||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 882: | Line 1,209: | ||
<div class="lightbox" id="exp22"> | <div class="lightbox" id="exp22"> | ||
<figure> | <figure> | ||
| − | <a href="# exp22" class="closemsg"></a> | + | <a href="#exp22" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U></br></br> | + | <U> Aim:</U>We want to check if our bacteria have produced enought ligated plasmid. </br></br> |
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
<U>Materials:</U></br> | <U>Materials:</U></br> | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| + | Spectro : Ultrospec 3100 pro-Amersham Bioscience</br> | ||
| + | Vol DNA = 2 l</br> | ||
| + | Vol TE buffer= 498 µl</br> | ||
| + | |||
| + | Dilution = 1/250</br> | ||
| + | In a quartz cuvette ( Path Length= 1 cm):</br> | ||
| + | - 1 ml of buffer TE</br> | ||
| + | - use 2 µl DNA in 998 µl of TE for the dilution</br> | ||
| + | Analysis to = 260 nm</br> | ||
| + | Blank on TE1X </br> | ||
| + | |||
<U>Results:</U></br></br> | <U>Results:</U></br></br> | ||
| + | <table> | ||
| + | <thead> | ||
| + | <tr> | ||
| + | <th>λ= 260 nm</th> | ||
| + | <th>Colony (1:1)</th> | ||
| + | <th>Colony (1:2)</th> | ||
| + | <th>Colony (2:1)</th> | ||
| + | <th>Colony (2:2)</th> | ||
| + | </tr> | ||
| + | </thead> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td><strong><p>Cfinale></p></strong></td> | ||
| + | <td>110 ng/µl</td> | ||
| + | <td>290 ng/µl</td> | ||
| + | <td>110 ng/µl</td> | ||
| + | <td>70 ng/µl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>Cfinale2></p></strong></td> | ||
| + | <td>5 500 ng/µl</td> | ||
| + | <td>14 500 ng/µl</td> | ||
| + | <td>5 500 ng/µl</td> | ||
| + | <td>3 500 ng/µl</td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | <center>Table 46</center> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 898: | Line 1,263: | ||
<div class="lightbox" id="exp23"> | <div class="lightbox" id="exp23"> | ||
<figure> | <figure> | ||
| − | <a href="# exp23" class="closemsg"></a> | + | <a href="#exp23" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U></br></br> | + | <U> Aim:</U>: We want to check if the plasmid has an insert.</br></br> |
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
<U>Materials:</U></br> | <U>Materials:</U></br> | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | < | + | <table> |
| + | <thead> | ||
| + | <tr> | ||
| + | <th></th> | ||
| + | <th>Colony (1:1)</th> | ||
| + | <th>Colony (1:2)</th> | ||
| + | <th>Colony (2:1)</th> | ||
| + | <th>Colony (2:2)</th> | ||
| + | </tr> | ||
| + | </thead> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td><strong><p>DNA (1 µg)</p></strong></td> | ||
| + | <td>9.1 µl</td> | ||
| + | <td>3.4 µl</td> | ||
| + | <td>9.1 µl</td> | ||
| + | <td>14.3µl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>XbaI</p></strong></td> | ||
| + | <td>1 µl</td> | ||
| + | <td>1 µl</td> | ||
| + | <td>1 µl</td> | ||
| + | <td>1 µl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>HindIII</p></strong></td> | ||
| + | <td>1 µl</td> | ||
| + | <td>1 µl</td> | ||
| + | <td>1 µl</td> | ||
| + | <td>1 µl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>H20</p></strong></td> | ||
| + | <td>15.9 µl</td> | ||
| + | <td>21.6 µl</td> | ||
| + | <td>15.9 µl</td> | ||
| + | <td>10.7 µl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>Buffer 10X</p></strong></td> | ||
| + | <td>3 µl</td> | ||
| + | <td>3 µl</td> | ||
| + | <td>3 µl</td> | ||
| + | <td>3 µl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>Buffer 10X</p></strong></td> | ||
| + | <td>30 µl</td> | ||
| + | <td>30 µl</td> | ||
| + | <td>30 µl</td> | ||
| + | <td>30 µl</td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | <center>Table 47</center></br></br> | ||
| + | 1. Add all reagents in a 1.5 ml eppendorf </br> | ||
| + | 2. Let the digestion proceed during 1h30 at 37 °C and incubate 10 min at 65 °C</br> | ||
| + | 3. For the reagent volumes, refer to the table</br> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 914: | Line 1,336: | ||
<div class="lightbox" id="exp24"> | <div class="lightbox" id="exp24"> | ||
<figure> | <figure> | ||
| − | <a href="# exp24" class="closemsg"></a> | + | <a href="#exp24" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U></br></br> | + | <U> Aim:</U>We want to check if the plasmid has an insert. </br></br> |
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
| − | |||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | <U>Results:</U></br></br> | + | <table> |
| + | <thead> | ||
| + | <tr> | ||
| + | <th></th> | ||
| + | <th>Lanes</th> | ||
| + | <th>L1</th> | ||
| + | <th>L2</th> | ||
| + | <th>L3</th> | ||
| + | <th>L4</th> | ||
| + | <th>L5</th> | ||
| + | <th>L6</th> | ||
| + | <th>L7</th> | ||
| + | <th>L8</th> | ||
| + | <th>L9</th> | ||
| + | <th>L10</th> | ||
| + | </tr> | ||
| + | </thead> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td><strong><p>Name</p></strong></td> | ||
| + | <td>Mark weight</td> | ||
| + | <td></td> | ||
| + | <td>pET43.1a(+) X/H</td> | ||
| + | <td></td> | ||
| + | <td>C2 (1:1)</td> | ||
| + | <td></td> | ||
| + | <td>C2 (1:2)</td> | ||
| + | <td></td> | ||
| + | <td>C2 (2:1)</td> | ||
| + | <td></td> | ||
| + | <td>C2 (2:2)</td> | ||
| + | <td>pET43.1a(+) uncut</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>DNA (µl)</p></strong></td> | ||
| + | <td>6</td> | ||
| + | <td></td> | ||
| + | <td>30</td> | ||
| + | <td></td> | ||
| + | <td>30</td> | ||
| + | <td></td> | ||
| + | <td>30</td> | ||
| + | <td></td> | ||
| + | <td>30</td> | ||
| + | <td></td> | ||
| + | <td>30</td> | ||
| + | <td>30</td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | <center>Table 48</center></br></br> | ||
| + | FIGURE </br></br> | ||
| + | <U>Results:</U></br></br> | ||
| + | After the elctrophoresis, we notice that the recombinating plasmid seems to contain two inserts. Indeed, the band corresponds to a size between 1 00 and 1 500 bp.</br> | ||
| + | We decided to digest our double insert with XbaI and SpeI to split it. </br> | ||
| + | We also digested a pET43.1with XbaI and SpeI to fit it. </br> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 930: | Line 1,405: | ||
<div class="lightbox" id="exp25"> | <div class="lightbox" id="exp25"> | ||
<figure> | <figure> | ||
| − | <a href="# exp25" class="closemsg"></a> | + | <a href="#exp25" class="closemsg"></a> |
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U></br></br> | + | <U> Aim:</U>We observed that two inserts were present in our gels, we therefore want to verify that the second heavier one is not as a result illegitimate ligation of XbaI/HindIII. There is a SpeI site in the neighboring sequences. Therefore, we want to split the insert to have just one insert.</br></br> |
<U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
<U>What we did in the lab:</U></br> | <U>What we did in the lab:</U></br> | ||
<U>Materials:</U></br> | <U>Materials:</U></br> | ||
<U>Method:</U></br> | <U>Method:</U></br> | ||
| − | <U>Results:</U></br></br> | + | 1. For volumes, refer to the next table :</br> |
| + | |||
| + | <table> | ||
| + | <thead> | ||
| + | <tr> | ||
| + | <th></th> | ||
| + | <th>C2.1.1 (110 ng/µl)</th> | ||
| + | <th>C2.1.2 (290 ng/µl)</th> | ||
| + | <th>pET43.1a(+)</th> | ||
| + | </tr> | ||
| + | </thead> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td><strong><p>Vol DNA (3 µl)</p></strong></td> | ||
| + | <td>27.3 µl</td> | ||
| + | <td>10.3 µl</td> | ||
| + | <td>2.5 µl</td> | ||
| + | |||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>Vol SepI (µl)</p></strong></td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>Vol XbaI (µl)</p></strong></td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | <td>1</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>H20 10X (µl)</p></strong></td> | ||
| + | <td>6.7</td> | ||
| + | <td>23.7</td> | ||
| + | <td>31.5</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>Vol Buffer CutSmart 10X (µl)</p></strong></td> | ||
| + | <td>4</td> | ||
| + | <td>4</td> | ||
| + | <td>4</td> | ||
| + | </tr> | ||
| + | <td><strong><p>Vol TOTAL (µl)</p></strong></td> | ||
| + | <td>40</td> | ||
| + | <td>40</td> | ||
| + | <td>40</td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | <center>Table 49</center></br></br> | ||
| + | 2. After 1H of digestion, we added 1 µl of each enzyme and let digest 30 more minutes. | ||
| + | </p> | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </div> | ||
| + | |||
| + | <div class="lightbox" id="exp26"> | ||
| + | <figure> | ||
| + | <a href="#exp26" class="closemsg"></a> | ||
| + | <figcaption> | ||
| + | <p> | ||
| + | <U> Aim:</U>We want to check if we succeeded in splitting the twinned insert.</br></br> | ||
| + | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
| + | <U>What we did in the lab:</U></br> | ||
| + | <U>Method:</U></br> | ||
| + | 1. To deposit volums, refer to this table :</br> | ||
| + | <table> | ||
| + | <thead> | ||
| + | <tr> | ||
| + | <th>Lines</th> | ||
| + | <th>L1</th> | ||
| + | <th>L2</th> | ||
| + | <th>L3</th> | ||
| + | <th>L4</th> | ||
| + | <th>L5</th> | ||
| + | <th>L6</th> | ||
| + | <th>L7</th> | ||
| + | </tr> | ||
| + | </thead> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td><strong><p>Name</p></strong></td> | ||
| + | <td>Mark weight</td> | ||
| + | <td></td> | ||
| + | <td>pET43.1 X/S</td> | ||
| + | <td></td> | ||
| + | <td>C2 (1:1)</td> | ||
| + | <td></td> | ||
| + | <td>C2 (1:2)</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>DNA (µl)</p></strong></td> | ||
| + | <td>10</td> | ||
| + | <td></td> | ||
| + | <td>40</td> | ||
| + | <td></td> | ||
| + | <td>40</td> | ||
| + | <td></td> | ||
| + | <td>40</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>H20 (µl)</p></strong></td> | ||
| + | <td>0</td> | ||
| + | <td></td> | ||
| + | <td>0</td> | ||
| + | <td></td> | ||
| + | <td>0</td> | ||
| + | <td></td> | ||
| + | <td>40</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><strong><p>H20 (µl)</p></strong></td> | ||
| + | <td>10</td> | ||
| + | <td></td> | ||
| + | <td>10</td> | ||
| + | <td></td> | ||
| + | <td>10</td> | ||
| + | <td></td> | ||
| + | <td>10</td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | <center>Table 50</center></br></br> | ||
| + | <U>Results:</U>The digestion does not work, we did not succeed in splitting the twinned insert so we decided to keep it and to express the protein with the double insert, hoping that the stop codon at the end of one of the inserts will be ennough. We will also sequence the plasmid to verify the orientation, and validity of our twinned insert hypothesis.</br></br> | ||
| + | </p> | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | <div class="lightbox" id="exp26"> | ||
| + | <figure> | ||
| + | <a href="#exp26" class="closemsg"></a> | ||
| + | <figcaption> | ||
| + | <p> | ||
| + | <U> Aim:</U>In this part we wantant to proceed with the expression of our fusion protein containing the R5-CBD-BPA. To do this we need a cell line having a T7 RNA polymerase. The cells we chose were BL21De3 pLys S. These contain the T7 phage producing the T7 RNA polymerase, and lysozyme, which will inhibit the T7 RNA polymrase and help control the expression better in case our protein is toxic to the cells, and secondly in case the cells escape our control they will not be able to survive in nature, as they will lyse over time.</br></br> | ||
| + | <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br> | ||
| + | <U>What we did in the lab:</U></br> | ||
| + | <U>Materials:</U></br>BL21De3 competent cells, SOC media, 42 °C waterbath.</br> | ||
| + | <U>Method:</U></br> | ||
| + | (I) : 50 µl of bacteria + 5 µl of pET43.1-C2 1.1</br> | ||
| + | (II) : 50 µl of bacteria + 5 µl of pET43.1-C2 1.2</br> | ||
| + | (III) : 50 µl of bacteria + 5 µl of pUC</br> | ||
| + | (IV) : 50 µl of bacteria + 5 µl of CT (plasmid given with the bacteria)</br></br></br> | ||
| + | After heat shock at 42°C, the cells were allowed to recover by growing at 37°C for 40 minutes in 150 µl of SOC, then the 200 µl of each sample were spread on a petridish with carbenicillin and grown at 37 °C for one night.</br></br> | ||
| + | |||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
