Difference between revisions of "Team:Pasteur Paris/Microbiology week14"
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<p><h3><B>September 8, 2016:</B></h3></p> | <p><h3><B>September 8, 2016:</B></h3></p> | ||
<p> | <p> | ||
| − | <a href="#exp3"><h4> 238. Protein gel on SDS- | + | <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> | <a href="#exp4"><h4> 239. Harvest the culture with Miniprep </h4></a></br> | ||
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<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> Produce our protein in BL21(DE3) competent cells, the production is induced with IPTG once it reaches an | + | <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> 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>What we did in the lab:</U></br> | ||
<U>Materials:</U></br> | <U>Materials:</U></br> | ||
• Two 2 l erlenmeyers</br> | • Two 2 l erlenmeyers</br> | ||
| − | • LB ( | + | • LB (Luria broth)</br> |
• IPTG (0.1 M)</br> | • IPTG (0.1 M)</br> | ||
• Precultures in 25 ml erlenmeyers</br> | • Precultures in 25 ml erlenmeyers</br> | ||
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• Shaking incubator (INFORS HT)</br> | • Shaking incubator (INFORS HT)</br> | ||
• Centrifuge</br> | • Centrifuge</br> | ||
| − | • Buffer A (50 mM | + | • Buffer A (Tris-Cl 50 mM pH 7.4, NaCl 150 mM)</br> </br> |
<U>Method:</U></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.< | + | 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.< | + | 2. Once warmed, add 12.5 ml of preculture in each erlenmeyer.<br/> |
| − | 3. Let grow in the shaking incubator.< | + | 3. Let grow in the shaking incubator.<br/> |
4. Measure the absorbance with the UV spectrophotometer every 30 minutes.</br> </br> | 4. Measure the absorbance with the UV spectrophotometer every 30 minutes.</br> </br> | ||
<table> | <table> | ||
| − | <caption align="bottom" align="center"><i><p> Table | + | <caption align="bottom" align="center"><i><p> <U>Table 141 :</U> Optical Density </p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
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</table><br/> | </table><br/> | ||
| − | 5. Add IPTG to reach a concentration of 0. | + | 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 | + | 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> | 7. Let induce overnight in the shaking incubator at 15°C at 150 rpm</br> | ||
| − | 8. The day after, measure the OD and store the measure pelleted at -20°C.</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> | 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/> | <br/><br/><br/> | ||
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<U>Materials:</U></br> | <U>Materials:</U></br> | ||
• Fast Purification Liquid Chromatography </br> | • Fast Purification Liquid Chromatography </br> | ||
| − | • Chaotropic reagent (Guanidinium 6 M) </br> | + | • Chaotropic reagent (Guanidinium HCL 6 M) </br> |
• EDTA 0,1 M </br> | • EDTA 0,1 M </br> | ||
• PMSF (100 mM) </br> | • PMSF (100 mM) </br> | ||
• Ni<sup>2+</sup> solution (100 mM) </br> | • Ni<sup>2+</sup> solution (100 mM) </br> | ||
• Centrifuge </br> | • Centrifuge </br> | ||
| − | • Buffer A (50 mM | + | • Buffer A (Tris 50 mM pH 7.4, NaCl 150 mM) </br> |
| − | • Buffer B (50 mM | + | • Buffer B (Tris 50 mM pH 7.4, NaCl 150 mM, Imidazole 250 mM) </br> </br> |
<U>Method:</U></br> | <U>Method:</U></br> | ||
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3. Put the column on the FPLC, clean the FPLC with water and then fill it with buffer A. </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> | 4. Sonicate the sample three times one minute at 60%, wait 90 seconds between each sonication. </br> | ||
| − | 5. Centrifuge 25 min at 16000g (rotor JA 25.50) </br> | + | 5. Centrifuge 25 min at 16000g (rotor Beckman JA 25.50) </br> |
6. Inject your sample in the FPLC </br> | 6. Inject your sample in the FPLC </br> | ||
7. Get back several samples: </br> | 7. Get back several samples: </br> | ||
| − | & | + |   → C: Crude extract : before centrigugation </br> |
| − | & | + |   → P: Pellet </br> |
| − | & | + |   → SN: Supernatant </br> |
| − | & | + |   → F: Flow through (unfixed proteins) </br> |
| − | & | + |   → W: Wash 5% of buffer B </br> |
| − | & | + |   → Fractions (depending on the gradient) </br> |
<br/><br/><br/> | <br/><br/><br/> | ||
</p> | </p> | ||
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<U>Material: </U> | <U>Material: </U> | ||
| − | • SDS- | + | • SDS-PAGE chamber <br/> |
| − | • SDS- | + | • SDS-PAGE gel 4-15% gradient (BIORAD) <br/> |
| − | • Protein migration buffer <br/> | + | • Protein migration buffer TGS 20X <br/> |
| − | • Protein ladder <br/> | + | • Protein ladder PAGE Ruler (Thermofisher)<br/> |
• Laemmli 2X <br/> | • Laemmli 2X <br/> | ||
• Coomassie Blue <br/> | • Coomassie Blue <br/> | ||
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<U>Method:</U><br/> | <U>Method:</U><br/> | ||
| − | 1. In nine 1.5 ml | + | 1. In nine 1.5 ml Eppendorf, put 20 µl of a sample and 20 µl of Laemmli 2X. <br/> |
2. Let denaturate the proteins 5 minutes at 95°C. <br/> | 2. Let denaturate the proteins 5 minutes at 95°C. <br/> | ||
| − | 3. Place the gel into the | + | 3. Place the gel into the chamber and fill it with migration buffer. <br/> |
| − | 4. Follow the next deposit table: <br/> | + | 4. Follow the next deposit table: <br/><br/> |
| + | |||
| + |   Fraction 13 | Fraction 15 | Fraction 17 | Fraction 19 | Fraction 21 | Fraction 23 | Fraction 25 | Fraction 27 | Protein gene ruler (8 µl)</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/> | <br/><br/><br/> | ||
</p> | </p> | ||
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<U>Method:</U><br/> | <U>Method:</U><br/> | ||
| − | 1. The protocol in step 1 ask for spinning at | + | 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 µl <br/> | 2. Follow QIAGEN kit steps. Final volume: 100 µl <br/> | ||
<br/><br/><br/> | <br/><br/><br/> | ||
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<U>Results:</U><br/> | <U>Results:</U><br/> | ||
<table> | <table> | ||
| − | <caption align="bottom" align="center"><i><p>Table | + | <caption align="bottom" align="center"><i><p><U>Table 142 :</U> Absorbance</p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
