Difference between revisions of "Team:Pasteur Paris/Microbiology week7"
| (4 intermediate revisions by 3 users not shown) | |||
| Line 255: | Line 255: | ||
<p> | <p> | ||
<U> Aim:</U> Check if our protein expression works. <br/> <br/> | <U> Aim:</U> Check if our protein expression works. <br/> <br/> | ||
| − | + | ||
<U>Results</U><br/> | <U>Results</U><br/> | ||
C2 1.1 (+)iPTG seems to have a new band around 30 kDa. However, C2 1.1 (+)iPTG and C2 1.2 (+)iPTG show a band at 70 kDa. As we have two inserts our plasmid it seems to be a double protein but it will not be efficient. So we decided to restart the induction | C2 1.1 (+)iPTG seems to have a new band around 30 kDa. However, C2 1.1 (+)iPTG and C2 1.2 (+)iPTG show a band at 70 kDa. As we have two inserts our plasmid it seems to be a double protein but it will not be efficient. So we decided to restart the induction | ||
| Line 273: | Line 273: | ||
<p> | <p> | ||
<U> Aim:</U> Do another culture of BL21DE3 to compare it to our previous one. <br/><br/> | <U> Aim:</U> Do another culture of BL21DE3 to compare it to our previous one. <br/><br/> | ||
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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/> | ||
| − | • Microbiology equipment < | + | • Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) |
• Culture of BL21DE3 <br/> | • Culture of BL21DE3 <br/> | ||
• Shaking incubator <br/> | • Shaking incubator <br/> | ||
| Line 285: | Line 285: | ||
2. Measure the concentration of the cultures at several times by absorbance at 600 nm: <br/> | 2. Measure the concentration of the cultures at several times by absorbance at 600 nm: <br/> | ||
<table> | <table> | ||
| − | <caption align="bottom" align="center">Table | + | <caption align="bottom" align="center"><i><p> <U>Table 55</U></p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 320: | Line 320: | ||
5. Measure the OD<sub>600</sub> : <br/> | 5. Measure the OD<sub>600</sub> : <br/> | ||
<table> | <table> | ||
| − | <caption align="bottom" align="center">Table | + | <caption align="bottom" align="center"><i><p> <U>Table 56</U></p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 358: | Line 358: | ||
<p> | <p> | ||
<U> Aim:</U> Do another culture of BL21DE3 to compare it to our previous one. <br/> <br/> | <U> Aim:</U> Do another culture of BL21DE3 to compare it to our previous one. <br/> <br/> | ||
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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/> | ||
| − | • Microbiology equipment < | + | • Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) |
• Culture of BL21DE3 <br/> | • Culture of BL21DE3 <br/> | ||
• Shaking incubator <br/> | • Shaking incubator <br/> | ||
| Line 386: | Line 386: | ||
<p> | <p> | ||
<U> Aim:</U> Get back the proteins produced by the bacteria. <br/> <br/> | <U> Aim:</U> Get back the proteins produced by the bacteria. <br/> <br/> | ||
| − | + | ||
<U> What we did in the lab </U><br/> | <U> What we did in the lab </U><br/> | ||
| − | • | + | • Lysis buffer B PER (Pierce)<br/> |
| − | • | + | • Bacteria pelleted <br/> |
• Laemmli 2X<br/> | • Laemmli 2X<br/> | ||
• 1.5 ml Eppendorf | • 1.5 ml Eppendorf | ||
| Line 396: | Line 396: | ||
1. Dilution to reach an OD<sub>600 nm</sub> of 10 : <br/> | 1. Dilution to reach an OD<sub>600 nm</sub> of 10 : <br/> | ||
<table> | <table> | ||
| − | <caption align="bottom" align="center">Table | + | <caption align="bottom" align="center"><i><p> <U>Table 57</U></p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 446: | Line 446: | ||
<p> | <p> | ||
<U> Aim:</U> Check if our protein has been produced (weight around 30 kDa). <br/> <br/> | <U> Aim:</U> Check if our protein has been produced (weight around 30 kDa). <br/> <br/> | ||
| − | + | ||
<U> Method </U><br/> | <U> Method </U><br/> | ||
Follow the deposit table : <br/> | Follow the deposit table : <br/> | ||
<table> | <table> | ||
| − | <caption align="bottom" align="center">Table | + | <caption align="bottom" align="center"><i><p> <U>Table 58</U></p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 480: | Line 480: | ||
<U> Results </U><br/> | <U> Results </U><br/> | ||
We did not observe at 30 kDa band for the induced cultures. <br/> | We did not observe at 30 kDa band for the induced cultures. <br/> | ||
| − | We notice a 70 kDa band when iPTG has been added (L5 – L9). This band is darker for the | + | We notice a 70 kDa band when iPTG has been added (L5 – L9). This band is darker for the C2 1.2 colony induced the whole night at 0.3 mM. <br/> |
We hypothesize that our protein is a fusion of the two proteins coded by the twinned insert. <br/> | We hypothesize that our protein is a fusion of the two proteins coded by the twinned insert. <br/> | ||
We will send our recombinant plasmid to sequencing to check the ligation. <br/> | We will send our recombinant plasmid to sequencing to check the ligation. <br/> | ||
| Line 498: | Line 498: | ||
<p> | <p> | ||
<U> Aim:</U> Check if the Histag works and if our protein has really been produce. <br/> <br/> | <U> Aim:</U> Check if the Histag works and if our protein has really been produce. <br/> <br/> | ||
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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> Materials </U><br/> | <U> Materials </U><br/> | ||
• Lysis buffer B PER <br/> | • Lysis buffer B PER <br/> | ||
| − | • Residue of the culture C2 1.1 and C2 1.2 (stored in 50 ml | + | • Residue of the culture C2 1.1 and C2 1.2 (stored in 50 ml Falcon at -20°C) <br/> |
• Protease inhibition PMSF at 100 mM <br/> | • Protease inhibition PMSF at 100 mM <br/> | ||
| + | • Laemli SDS buffer 2X</br> | ||
• Tris at 1 M <br/> | • Tris at 1 M <br/> | ||
• NaCl at 5 M <br/> | • NaCl at 5 M <br/> | ||
• Imidazole at 1.5 M <br/><br/> | • Imidazole at 1.5 M <br/><br/> | ||
<U> Method </U><br/> | <U> Method </U><br/> | ||
| + | Adapt the protocol based on the culture volume. In this case for a small volume, we used B-PER reagent to lyse the cells.</br> | ||
1. Lysis of bacteria : <br/> | 1. Lysis of bacteria : <br/> | ||
| − |   1.a For C2 1.1 ( | + |   1.a For C2 1.1 (21 ml), add 1 ml of lysis buffer <br/> |
| − |   1.b For C2 1.2 ( | + |   1.b For C2 1.2 (19 ml), add 1 ml of lysis buffer <br/> |
2. Let lysate during at least 5 minutes on ice <br/> | 2. Let lysate during at least 5 minutes on ice <br/> | ||
3. Add 1.5 μl of PMSF to reach a concentration of 15 μM in each sample <br/> | 3. Add 1.5 μl of PMSF to reach a concentration of 15 μM in each sample <br/> | ||
4. Preparation of buffer A : in a 500 ml bottle, put 25 ml of Tris and 15 ml of NaCl <br/> | 4. Preparation of buffer A : in a 500 ml bottle, put 25 ml of Tris and 15 ml of NaCl <br/> | ||
5. Measure the pH and correct it by adding NaOH or HCl to reach 7.4. In our case, the solution was too acid so we add droplets of NaOH <br/> | 5. Measure the pH and correct it by adding NaOH or HCl to reach 7.4. In our case, the solution was too acid so we add droplets of NaOH <br/> | ||
| − | 6. Fill the bottle with osmosed Millipore water to reach a final concentration of 50 mM for Tris and 150 mM for NaCl <br/> | + | 6. Fill the bottle with reverse-osmosed MilliQ (Millipore) water to reach a final concentration of 50 mM for Tris and 150 mM for NaCl <br/> |
7. Preparation of buffer B (elution) : in a 500 ml bottle, put 25 ml of Tris, 15ml of NaCl and 125 ml of imidazole <br/> | 7. Preparation of buffer B (elution) : in a 500 ml bottle, put 25 ml of Tris, 15ml of NaCl and 125 ml of imidazole <br/> | ||
| − | 8. Correct the pH to reach 7.4 and fill with water to reach a final concentration of 50 mM for Tris, 150 mM for NaCl and 250 mM of imidazole <br/> | + | 8. Correct the pH to reach 7.4 and fill with reverse-osmosed MilliQ water to reach a final concentration of 50 mM for Tris, 150 mM for NaCl and 250 mM of imidazole <br/> |
| − | 9. In a clean 50 ml | + | 9. In a clean 50 ml Falcon, put the samples (one for C2 1.1 and one for C2 1.2) and add 10 ml of buffer A <br/> |
10. As our samples were too sticky we sonicate them during a few seconds <br/> | 10. As our samples were too sticky we sonicate them during a few seconds <br/> | ||
11. Centrifuge the samples 20 minutes at 16000 RPM (30966 g) <br/> | 11. Centrifuge the samples 20 minutes at 16000 RPM (30966 g) <br/> | ||
| − | 12. | + | 12. Filter the buffers to eliminate impurities on a funnel equipped with 0.45 µm Nylon filter (Whatman)<br/> |
| − | 13. The supernatant | + | 13. The supernatant is put in a clean 50 ml Falcon <br/> |
| − | 14. Purification of protein with AKTA FPLC from GE Healthcare <br/> | + | 14. Purification of protein with AKTA FPLC from GE Healthcare, following the Abs<sub>280 nm</sub> <br/> |
| − | 15. All the tubes are | + | 15. Collect 1 ml Fraction |
| + | 15. All the fraction tubes are stored at 4°C <br/><br/> | ||
<U> Results </U><br/> | <U> Results </U><br/> | ||
<center><img src=" ; alt=""></center> | <center><img src=" ; alt=""></center> | ||
<center>Figure 3</center> | <center>Figure 3</center> | ||
| + | Perform a SDS-PAGE separation of the fractions collected, as well as supernatant of crude lysate and pellet.</br> | ||
The protein is in the supernatant and not in the pellet. <br/> | The protein is in the supernatant and not in the pellet. <br/> | ||
| − | After the purification, we | + | After the purification, we noticed that some samples contain a protein that was hang onto the Nickel column. <br/> |
| − | We decided to do | + | We decided to do an SDS-PAGE gel to analyse the size of this protein |
<br/><br/><br/> | <br/><br/><br/> | ||
</p> | </p> | ||
| Line 543: | Line 547: | ||
<figcaption> | <figcaption> | ||
<p> | <p> | ||
| − | <U> Aim:</U> Do an SDS-PAGE gel to | + | <U> Aim:</U> Do an SDS-PAGE gel to verify the purification our proteins. <br/> <br/> |
| − | + | ||
| − | + | ||
• Lysis buffer B PER <br/> | • Lysis buffer B PER <br/> | ||
| − | • Residue of the culture C2 1.1 and C2 1.2 (stored in 50 ml | + | • Polyacrylamide precast gel for mini Protean II (Biorad) systems 4-15% gradient in TGS buffer (Tris-Glycine SDS)</br> |
| + | • Residue of the culture C2 1.1 and C2 1.2 (stored in 50 ml Falcon at -20°C) <br/> | ||
• Protease inhibition PMSF at 100 mM <br/> | • Protease inhibition PMSF at 100 mM <br/> | ||
• Tris at 1 M <br/> | • Tris at 1 M <br/> | ||
• NaCl at 5 M <br/> | • NaCl at 5 M <br/> | ||
| + | • Laemli SDS buffer 2X</br> | ||
• Imidazole at 1.5 M <br/><br/> | • Imidazole at 1.5 M <br/><br/> | ||
<U> Method </U><br/> | <U> Method </U><br/> | ||
Each sample has to be diluted with Laemmli 2X. <br/> | Each sample has to be diluted with Laemmli 2X. <br/> | ||
| − | 1. Take 20 μl of the sample and 20 μl of Laemmli 2X and put | + | 1. Take 20 μl of the sample and 20 μl of Laemmli 2X and put the heating block for 5 minutes at 95°C to denaturate the protein <br/> |
2. Follow the deposit table : | 2. Follow the deposit table : | ||
<table> | <table> | ||
| − | <caption align="bottom" align="center">Table | + | <caption align="bottom" align="center"><i><p> <U>Table 59</U></p></i></caption> |
<thead> | <thead> | ||
<tr> | <tr> | ||
| Line 588: | Line 593: | ||
</table><br/> | </table><br/> | ||
3. Migration at 130 V for 45 minutes <br/> | 3. Migration at 130 V for 45 minutes <br/> | ||
| − | 4. Do 3 washes of 5 with distilled water and then 50 minutes of coloration with Coomasie blue diluted 1⁄5 <br/><br/> | + | 4. Do 3 washes of 5 min with distilled water and then 50 minutes of coloration with Coomasie blue diluted 1⁄5 <br/><br/> |
<U> Results </U><br/> | <U> Results </U><br/> | ||
A band located at 70 kDa appears for the sample 29 | A band located at 70 kDa appears for the sample 29 | ||
| Line 606: | Line 611: | ||
<p> | <p> | ||
<U> Aim:</U> Check if our fusion protein is efficient for silification. <br/> <br/> | <U> Aim:</U> Check if our fusion protein is efficient for silification. <br/> <br/> | ||
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/2/2a/Protocol-silification-assay_Pasteur_Paris2016.pdf">link</a><br/><br/> |
<U> Materials </U><br/> | <U> Materials </U><br/> | ||
• Biochemical equipment : pipettes, cones, Eppendorf tubes, … <br/> | • Biochemical equipment : pipettes, cones, Eppendorf tubes, … <br/> | ||
| − | • TEOS <br/> | + | • TEOS (Tetraethyl Orthosilicate, Sigma)<br/> |
• HCl 1 M <br/> | • HCl 1 M <br/> | ||
• Ulltrospec 3000 pro <br/> | • Ulltrospec 3000 pro <br/> | ||
| Line 619: | Line 624: | ||
1.For fractions 27 to 31 : <br/> | 1.For fractions 27 to 31 : <br/> | ||
  1.a In a 50 ml Falcon, put 10 μl of our sample of protein in 10 ml of TEOS. <br/> |   1.a In a 50 ml Falcon, put 10 μl of our sample of protein in 10 ml of TEOS. <br/> | ||
| − |   1.b In another 50 | + |   1.b In another 50 ml Falcon, put 10 μl of our sample of protein and 10 ml of water <br/> |
2.For the other fractions : <br/> | 2.For the other fractions : <br/> | ||
| − |   2.a Prepare a solution of (Si(CH)4+HCl) with 55.8 μl of TEOS and 1 ml of HCl at 1 mM <br/> | + |   2.a Prepare a solution of (Si(CH<sub>3</sub>O)<sub>4</sub>+HCl) with 55.8 μl of TEOS and 1 ml of HCl at 1 mM <br/> |
| − |   2.b In a 50 ml Falcon, put 50 μl of our purified protein with the solution of (Si(CH)<sub>4</sub>+HCl) and let it stand for | + |   2.b In a 50 ml Falcon, put 50 μl of our purified protein with the solution of (Si(CH<sub>3</sub>O)<sub>4</sub>+HCl) and let it stand for 4 minutes at room temperature <br/> |
  2.c Add 9 ml of buffer B <br/> |   2.c Add 9 ml of buffer B <br/> | ||
<U>Results</U><br/> | <U>Results</U><br/> | ||
| − | In spite of some impurities in the Fractions by the current purification method, and the difficulties to overcome cellulose in almost all | + | In spite of some impurities in the Fractions by the current purification method, and the difficulties to overcome cellulose in almost all commercially available FPLC purification columns, we decided to make more protein before testing because we did not have enough. <br/><br/> |
Hypothesis: for the concentration of our protein, 1 mg⁄ml correspond to 100 mM<br/><br/> | Hypothesis: for the concentration of our protein, 1 mg⁄ml correspond to 100 mM<br/><br/> | ||
The protein concentration was measured by its absorbance at 280 nm: A = 0.10400 <br/> | The protein concentration was measured by its absorbance at 280 nm: A = 0.10400 <br/> | ||
| − | We assumed to have an extinction coefficient of 1 OD 280 nm= 1 mg⁄ml protein <br/> | + | We assumed to have an extinction coefficient of 1 OD<sub>280 nm</sub>= 1 mg⁄ml protein <br/> |
Thus our [protein] = 0.104 mg⁄ml<br/> | Thus our [protein] = 0.104 mg⁄ml<br/> | ||
We need to produce more protein by the bacteria cells to reach the concentration of 1 mM which correspond to 70 mg⁄ml. | We need to produce more protein by the bacteria cells to reach the concentration of 1 mM which correspond to 70 mg⁄ml. | ||
| Line 647: | Line 652: | ||
<p> | <p> | ||
<U> Aim:</U> Make a preculture of BL21DE3 to increase the amount of bacteria available for innoculation later. <br/> <br/> | <U> Aim:</U> Make a preculture of BL21DE3 to increase the amount of bacteria available for innoculation later. <br/> <br/> | ||
| − | <U> Protocol:</U> follow in this <a href="https:// | + | <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> Materials </U><br/> | <U> Materials </U><br/> | ||
| − | • Microbiology equipment ( | + | • Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) |
| − | • | + | • carbenicillin at 50 mg⁄ml <br/> |
• LB <br/> | • LB <br/> | ||
• C2 1.2 colony <br/> | • C2 1.2 colony <br/> | ||
| Line 656: | Line 661: | ||
• Shaking incubator at 37 °C <br/><br/> | • Shaking incubator at 37 °C <br/><br/> | ||
<U> Method </U><br/> | <U> Method </U><br/> | ||
| − | 1. In two Erlenmeyers of 50 ml, put 25 ml of LB and 25 μl of | + | 1. In two Erlenmeyers of 50 ml, put 25 ml of LB and 25 μl of carbenicillin <br/> |
2. Add in each Erlenmeyer one colony of C2 1.2 from the plate stored at 4°C <br/> | 2. Add in each Erlenmeyer one colony of C2 1.2 from the plate stored at 4°C <br/> | ||
3. Put the two Erlenmeyers in the shaking incubator at 37°C and 150 rpm overnight. | 3. Put the two Erlenmeyers in the shaking incubator at 37°C and 150 rpm overnight. | ||
Latest revision as of 00:53, 20 October 2016
