Difference between revisions of "Team:Pasteur Paris/Microbiology week6"
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>Make a growth curve and induce the production of protein. We will start producing our protein in bacterial cells. In order to do that we need to have an idea of the growth profile of the cells with this construct inside. This will determine at what time we can induce the expression of our protein.</br></br> | + | <h6><U> Aim:</U></h6> Make a growth curve and induce the production of protein. We will start producing our protein in bacterial cells. In order to do that we need to have an idea of the growth profile of the cells with this construct inside. This will determine at what time we can induce the expression of our protein.</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <h6><U> Protocol:</U></h6> 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> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Materials:</U></ | + | <h6><U>Materials:</U></h6> |
| − | + | • Erlenmeyer flasks 250 ml capacity <br /> | |
| + | • IPTG (Iso-propyl β thio-galactopyranoside) 0.5 M <br /> | ||
| + | • LB media, carbenicillin at 50 mg/ml <br /> | ||
| + | • Shaking incubator (Infors HT) <br /> | ||
| + | • Spectrophotometer.</br></br> | ||
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
| − | 1. 2 x 250 ml Erlenmeyer, put 25 ml of LB and 25 | + | 1. 2 x 250 ml Erlenmeyer, put 25 ml of LB and 25 µl of carbenicillin at 50 mg/ml</br> |
2. then add in one colony of BL21DE3 pLys S with C2 1.2 and in the other flask add one with C 1.1</br> | 2. then add in one colony of BL21DE3 pLys S with C2 1.2 and in the other flask add one with C 1.1</br> | ||
3. put them 1 hour at 37°C and 180 RPM</br> | 3. put them 1 hour at 37°C and 180 RPM</br> | ||
4. Store the master plate (reference) at -4°C and make a copy plate grown at 37°C, then stored at 4°C.</br> | 4. Store the master plate (reference) at -4°C and make a copy plate grown at 37°C, then stored at 4°C.</br> | ||
| − | 5. Measure absorbance at | + | 5. Measure absorbance at 600 nm (Abs<sub>600 nm</sub>) to make a growth curve (Ultrospec 3100 pro, GE Lifesciences) using plain plastic cuvettes, 1 cm path length. |
</br></br> | </br></br> | ||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 52</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
<th>Time</th> | <th>Time</th> | ||
| − | <th>C2 1.1 | + | <th>C2 1.1 Abs<sub>600 nm</sub></th> |
| − | <th>C2 1.2 | + | <th>C2 1.2 Abs<sub>600 nm</sub></th> |
</tr> | </tr> | ||
| Line 280: | Line 291: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>13h59</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>13h59</p></strong></td> |
| − | <td>0.128</td> | + | <td align = "center"; valign = "center">0.128</td> |
| − | <td>0.044</td> | + | <td align = "center"; valign = "center">0.044</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>14h25 </strong></td> | + | <td align = "center"; valign = "center"><strong>14h25 </strong></td> |
| − | <td>0.168</td> | + | <td align = "center"; valign = "center">0.168</td> |
| − | <td>0.095</td> | + | <td align = "center"; valign = "center">0.095</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>14h50</strong></td> | + | <td align = "center"; valign = "center"><strong>14h50</strong></td> |
| − | <td>0.282</td> | + | <td align = "center"; valign = "center">0.282</td> |
| − | <td>0.188</td> | + | <td align = "center"; valign = "center">0.188</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>15h17</strong></td> | + | <td align = "center"; valign = "center"><strong>15h17</strong></td> |
| − | <td>0.387</td> | + | <td align = "center"; valign = "center">0.387</td> |
| − | <td>0.262</td> | + | <td align = "center"; valign = "center">0.262</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>15h32</strong></td> | + | <td align = "center"; valign = "center"><strong>15h32</strong></td> |
| − | <td>0.722</td> | + | <td align = "center"; valign = "center">0.722</td> |
| − | <td>0.620</td> | + | <td align = "center"; valign = "center">0.620</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong>16h07 </strong></td> | + | <td align = "center"; valign = "center"><strong>16h07 </strong></td> |
| − | <td></td> | + | <td align = "center"; valign = "center"></td> |
| − | <td> | + | <td align = "center"; valign = "center">0.675</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table | + | </table></br></br> |
| − | + | ||
| − | + | ||
1. When absorbance reached 0.7, we added IPTG at 0.5 mM to induce the production of protein</br> | 1. When absorbance reached 0.7, we added IPTG at 0.5 mM to induce the production of protein</br> | ||
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2. We let the induction proceed during 3 hours at 37°C and 180 RPM | 2. We let the induction proceed during 3 hours at 37°C and 180 RPM | ||
| − | Measure of absorbance: | + | Measure of absorbance: OD<sub>600 nm</sub> C2 1.1= 0.865, OD<sub>600 nm</sub> C2 1.2 =0.846</br></br> |
| − | <U>Results: </U>Our experiment failed because the agitation was stopped ! (Someone unfortunately either didn't restart the incubator shaking mode or programmed a small time on the shaker's timer by error). We pelleted 1 ml of each culture (3 min at 6800 g) and stored at -20°C. The Erlenmeyer content was pelleted too (5 min at 4500 RPM) and stored at -80°C. </U></br> | + | <h6><U>Results:</U></h6> Our experiment failed because the agitation was stopped ! (Someone unfortunately either didn't restart the incubator shaking mode or programmed a small time on the shaker's timer by error). We pelleted 1 ml of each culture (3 min at 6800 g) and stored at -20°C. The Erlenmeyer content was pelleted too (5 min at 4500 RPM) and stored at -80°C. </U></br> |
| − | In the meanwhile, during the growth curve experiment, we performed all the steps for the transformation of the new versions of our inserts synthesized | + | In the meanwhile, during the growth curve experiment, we performed all the steps for the transformation of the new versions of our inserts synthesized by IDT.</U></br> |
| − | We indeed decided to | + | We indeed decided to re-synthesized our inserts, such that it had a few improvements, namely that now it'll depend on the vector's promoter, the linker regions had less GC content, and were shortened. We will refer to these constructs as X.V2.<br /> |
| − | + | <br /><br /><br /> | |
| − | + | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> | + | <h6><U> Aim:</U></h6> After the digestion we dephosphorylated it to prevent that pET43.1 to religate without the insert.</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/c0/T--Pasteur_Paris--dephosphorylation_protocol.pdf">link</a></br></br><br /> |
| − | <U>What we did in the lab:</U></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
In a 1 ml Eppendorf, we put: </br> | In a 1 ml Eppendorf, we put: </br> | ||
| − | - 25 | + | - 25 µl of pET43.1 (3 µg/50 µl) </br> |
| − | - 2 | + | - 2 µl of buffer Cutsmart 10X </br> |
| − | - 1 | + | - 1 µl of SAP </br> |
| − | TOTAL = 28 | + | TOTAL = 28 µl </br> |
| − | </br> | + | </br><br /><br /> |
</p> | </p> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> | + | <h6><U> Aim:</U></h6>Integrate the inserts into the plasmid</br></br> |
| − | <U> Protocol:</U> follow | + | <h6><U> Protocol:</U></h6>follow in this <a href="https://static.igem.org/mediawiki/2016/a/ab/T--Pasteur_Paris--Restriction_digestion_protocol.pdf">link</a></br></br> |
| − | + | <br /><br /><br /> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>Purify the | + | <h6><U> Aim:</U></h6> Purify the digested inserts (C1/C2/B1/B2) and the plasmid before the ligation.</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <h6><U>Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a></br></br><br /> |
| − | <U>What we did in the lab:</U></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
| − | 1. make | + | 1. make a 0.7% agarose gel</br> |
2. do an electrophoresis following the deposit table</br></br> | 2. do an electrophoresis following the deposit table</br></br> | ||
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Gel mass: B1: 304 mg</br> B2: 382 mg</br> C1: 378 mg</br> C2: 451 mg</br> | Gel mass: B1: 304 mg</br> B2: 382 mg</br> C1: 378 mg</br> C2: 451 mg</br> | ||
| − | + | <br /><br /><br /> | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> | + | <h6><U> Aim:</U></h6> To Prepare our plasmids for the transformation.</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/6/6f/T--Pasteur_Paris--Ligation_protocol.pdf">link</a></br></br> |
| − | <U>What we did in the lab:</U></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
For each insert, we make two samples.</br></br> | For each insert, we make two samples.</br></br> | ||
| − | |||
| − | |||
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 53</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 420: | Line 427: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>DNA (200 ng)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>DNA (200 ng)</p></strong></td> |
| − | <td>4 | + | <td align = "center"; valign = "center">4 µl</td> |
| − | <td>4 | + | <td align = "center"; valign = "center">4 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Insert B1/B2/C1/C2 (version 2)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Insert B1/B2/C1/C2 (version 2)</p></strong></td> |
| − | <td>4 | + | <td align = "center"; valign = "center">4 µl</td> |
| − | <td>12 | + | <td align = "center"; valign = "center">12 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>T4 ligase</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>T4 ligase</p></strong></td> |
| − | <td>1 | + | <td align = "center"; valign = "center">1 µl</td> |
| − | <td>1 | + | <td align = "center"; valign = "center">1 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Buffer | + | <td align = "center"; valign = "center"><strong><p>Buffer 10X</p></strong></td> |
| − | <td>2 | + | <td align = "center"; valign = "center">2 µl</td> |
| − | <td>2 | + | <td align = "center"; valign = "center">2 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p> | + | <td align = "center"; valign = "center"><strong><p>H<sub>2</sub>0</p></strong></td> |
| − | <td>9 | + | <td align = "center"; valign = "center">9 µl</td> |
| − | <td>1 | + | <td align = "center"; valign = "center">1 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>TOTAL</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>TOTAL</p></strong></td> |
| − | <td>20 | + | <td align = "center"; valign = "center">20 µl</td> |
| − | <td>20 | + | <td align = "center"; valign = "center">20 µl</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table> | + | </table><br /><br /> |
| − | + | </br></br></br> | |
| − | </br></br></br> </p> | + | </p> |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> | + | <h6><U> Aim:</U></h6> Transform our plasmids into competent cells to amplify the DNA. </br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a></br></br><br /> |
| − | <U>What we did in the lab:</U></br> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
| − | Moreover, we spread the competent cells from July 7,2016 on petri | + | Moreover, we spread the competent cells from July 7,2016 on petri dishes to have new colonies.</br> |
| + | <br /><br /><br /> | ||
</p> | </p> | ||
</figcaption> | </figcaption> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> As our culture of BL21DE3 | + | <h6><U> Aim:</U></h6> As our culture of BL21DE3 didn’t work we redid exactly the same experiment as yesterday.</br></br> |
<table> | <table> | ||
| + | <caption align="bottom" align="center"><i><p> <U>Table 54</U></p></i></caption> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
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<tbody> | <tbody> | ||
<tr> | <tr> | ||
| − | <td><strong><p>DNA (200 ng)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>DNA (200 ng)</p></strong></td> |
| − | <td>4 | + | <td align = "center"; valign = "center">4 µl</td> |
| − | <td>4 | + | <td align = "center"; valign = "center">4 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Insert B1/B2/C1/C2 (version 2)</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>Insert B1/B2/C1/C2 (version 2)</p></strong></td> |
| − | <td>4 | + | <td align = "center"; valign = "center">4 µl</td> |
| − | <td>12 | + | <td align = "center"; valign = "center">12 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>T4 ligase</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>T4 ligase</p></strong></td> |
| − | <td>1 | + | <td align = "center"; valign = "center">1 µl</td> |
| − | <td>1 | + | <td align = "center"; valign = "center">1 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>Buffer | + | <td align = "center"; valign = "center"><strong><p>Buffer 10X</p></strong></td> |
| − | <td>2 | + | <td align = "center"; valign = "center">2 µl</td> |
| − | <td>2 | + | <td align = "center"; valign = "center">2 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p> | + | <td align = "center"; valign = "center"><strong><p>H<sub>2</sub>0</p></strong></td> |
| − | <td>9 | + | <td align = "center"; valign = "center">9 µl</td> |
| − | <td>1 | + | <td align = "center"; valign = "center">1 µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><strong><p>TOTAL</p></strong></td> | + | <td align = "center"; valign = "center"><strong><p>TOTAL</p></strong></td> |
| − | <td>20 | + | <td align = "center"; valign = "center">20 µl</td> |
| − | <td>20 | + | <td align = "center"; valign = "center">20 µl</td> |
</tr> | </tr> | ||
</tbody> | </tbody> | ||
| − | </table> | + | </table><br /><br /> |
| − | + | </br></br></br> | |
| − | </br></br></br> | + | |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
| Line 532: | Line 540: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>Check | + | <h6><U> Aim:</U></h6>Check if the bacteria that were induced by IPTG have produced the protein (weight = 30 kDa).</br></br> |
| − | < | + | |
| − | + | <h6><U>What we did in the lab:</U></h6></br> | |
| − | <U>Materials:</U></ | + | <h6><U>Materials:</U></h6> |
| − | + | • mini protean TGX 4-15% precast gels (BioRad)</br> | |
| − | + | • TGS Buffer 10X</br> | |
| − | + | • Electrophoresis chamber</br> | |
| − | + | • Samples of our culture </br></br> | |
| − | C2 1.1 (-) IPTG</br> | + | • C2 1.1 (-) IPTG</br> |
| − | C2 1.1 (+) IPTG</br> | + | • C2 1.1 (+) IPTG</br> |
| − | C2 1.2 (-) IPTG</br> | + | • C2 1.2 (-) IPTG</br> |
| − | C2 1.2 (+) IPTG</br> | + | • C2 1.2 (+) IPTG</br> |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
| − | 1. Put the gel in the | + | 1. Put the gel in the electrophoresis chamber, take off the green parts off the gel before</br> |
| − | 2. Fill the | + | 2. Fill the chamber with the buffer 1X</br> |
3. Follow the deposit table:</br> | 3. Follow the deposit table:</br> | ||
| − | L1: 8 | + | L1: 8 µl ladder protein Thermofisher (PAGE Ruler plus)</br> |
| − | L3: 17 | + | L3: 17 µl of C2 1.1 (-) IPTG</br> |
| − | L6: 17 | + | L6: 17 µl of C2 1.1 (+) IPTG</br> |
| − | L8: 17 | + | L8: 17 µl of C2 1.2 (-) IPTG</br> |
| − | L10: 17 | + | L10: 17 µl C2 1.2 (+) IPTG</br></br> |
In each eppendorg we added: </br> | In each eppendorg we added: </br> | ||
| − | - 10 | + | - 10 µl of protein solution</br> |
| − | - 10 | + | - 10 µl of laemli 2X</br> |
| − | Heat the samples at 95°C for 5 min to | + | Heat the samples at 95°C for 5 min to denature the protein</br> |
4. start of migration at 130 V</br> | 4. start of migration at 130 V</br> | ||
5. Stop the migration about one hour later</br> | 5. Stop the migration about one hour later</br> | ||
6. Open the plastic content to get back the gel</br> | 6. Open the plastic content to get back the gel</br> | ||
| − | 7. Wash three times five minutes in distilled | + | 7. Wash three times five minutes in distilled H<sub>2</sub>0</br> |
8. Fill a cuve with Bleu de Coomassie and let color the gel during 30 minutes. Let one hour more if required</br> | 8. Fill a cuve with Bleu de Coomassie and let color the gel during 30 minutes. Let one hour more if required</br> | ||
| − | 9. Wash distilled | + | 9. Wash distilled H<sub>2</sub>0 until the bands appear</br></br> |
| − | <U> Results:</U></ | + | <h6><U> Results:</U></h6> |
| − | It seems to have a slight band for C2 11 (+) IPTG that could be our protein but this band doesn’t appear in C2 1.2 (+) IPTG. We decided to let it the whole weekend in distilled | + | It seems to have a slight band for C2 11 (+) IPTG that could be our protein but this band doesn’t appear in C2 1.2 (+) IPTG. We decided to let it the whole weekend in distilled H<sub>2</sub>0 to analyze the bands.</br> |
| + | </br><br /><br /> | ||
</p> | </p> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U>get back the plasmid | + | <h6><U> Aim:</U></h6> To get back the plasmid from the cultures made on the July 11, 2016.</br></br> |
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <h6><U> Protocol:</U></h6> 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> | + | <h6><U>What we did in the lab:</U></h6></br> |
| − | <U>Method:</U></ | + | <h6><U>Method:</U></h6> |
| − | Once, the DNA | + | Once, the DNA has been pelleted let the Eppendorf open to evacuate the ethanol (all the week-end).</br> |
</p> | </p> | ||
</figcaption> | </figcaption> | ||
