Team:Pasteur Paris/Microbiology week7
83. Checking of the gel
84. Culture of BL21DE3
85. Continuation of the BL21DE3 cultures
86. Lysis of bacteria
87. Protein gel
88. Purification on nickel columns
89. SDS-PAGE gel
90. Silification tests
July 24, 2016:
Aim: Check if our protein expression works.
Results
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
Aim: Do another culture of BL21DE3 to compare it to our previous one.
Protocol: follow in this link
What we did in the lab
Materials
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• Culture of BL21DE3
• Shaking incubator
• 1.5 ml Eppendorfs
Method
1. Put the cultures in the shaking incubator at 37°C and 180 rpm at 10h07
2. Measure the concentration of the cultures at several times by absorbance at 600 nm:
| Times of measurement | C2 1.1 (Abs 600 nm) | C2 1.2 (Abs 600 nm) |
|---|---|---|
3h33 |
0.014 | 0.041 |
4h43 |
0.219 | 0.020 |
5h30 |
0.593 | 0.039 |
5h58 |
0.734 | 0.058 |
| Colonies | OD600 |
|---|---|
C2 1.1 |
0.928 |
C1 1.2 |
0.827 |
Aim: Do another culture of BL21DE3 to compare it to our previous one.
Protocol: follow in this link
What we did in the lab
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• Culture of BL21DE3
• Shaking incubator
• 1.5 ml Eppendorfs
Method
1. The sample induced during the night has an OD600 nm of 1.165 at 11h15.
2. The measurement is put in a 1.5 ml Eppendorf and stored at -20°C
3. Centrifuge 3 minutes at 7000 g and throw away the supernatent
4. All the culture is put in a 50 ml Falcon, centrifuged for 15 minutes at 4500 g.
5. Throw away the supernatant and store the Falcon at -20°C
Aim: Get back the proteins produced by the bacteria.
What we did in the lab
• Lysis buffer B PER (Pierce)
• Bacteria pelleted
• Laemmli 2X
• 1.5 ml Eppendorf
Method
1. Dilution to reach an OD600 nm of 10 :
| OD600 nm | Volume of lysis buffer to add (μl) | |
|---|---|---|
C2 1.1 (-)iPTG |
0.734 | 73.52 |
C2 1.1 (+)iPTG |
0.928 | 92.8 |
C2 1.2 (-)iPTG |
0.827 | 82.7 |
C2 1.2 (+)iPTG |
1.165 | 116.3 |
Aim: Check if our protein has been produced (weight around 30 kDa).
Method
Follow the deposit table :
| L1 | L2 | L3 | L4 | L5 | L6 | L7 | L8 | L9 |
|---|---|---|---|---|---|---|---|---|
| Ladder | Ø | 1.1(-) | Ø | 1.1(+) | Ø | 1.2(-) | Ø | 1.2(+) |
Aim: Check if the Histag works and if our protein has really been produce.
Protocol: follow in this link
Materials
• Lysis buffer B PER
• Residue of the culture C2 1.1 and C2 1.2 (stored in 50 ml Falcon at -20°C)
• Protease inhibition PMSF at 100 mM
• Laemli SDS buffer 2X
• Tris at 1 M
• NaCl at 5 M
• Imidazole at 1.5 M
Method
Adapt the protocol based on the culture volume. In this case for a small volume, we used B-PER reagent to lyse the cells.
1. Lysis of bacteria :
1.a For C2 1.1 (21 ml), add 1 ml of lysis buffer
1.b For C2 1.2 (19 ml), add 1 ml of lysis buffer
2. Let lysate during at least 5 minutes on ice
3. Add 1.5 μl of PMSF to reach a concentration of 15 μM in each sample
4. Preparation of buffer A : in a 500 ml bottle, put 25 ml of Tris and 15 ml of NaCl
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
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
7. Preparation of buffer B (elution) : in a 500 ml bottle, put 25 ml of Tris, 15ml of NaCl and 125 ml of imidazole
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
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
10. As our samples were too sticky we sonicate them during a few seconds
11. Centrifuge the samples 20 minutes at 16000 RPM (30966 g)
12. Filter the buffers to eliminate impurities on a funnel equipped with 0.45 µm Nylon filter (Whatman)
13. The supernatant is put in a clean 50 ml Falcon
14. Purification of protein with AKTA FPLC from GE Healthcare, following the Abs280 nm
15. Collect 1 ml Fraction
15. All the fraction tubes are stored at 4°C
Results
Aim: Do an SDS-PAGE gel to verify the purification our proteins.
• Lysis buffer B PER
• Polyacrylamide precast gel for mini Protean II (Biorad) systems 4-15% gradient in TGS buffer (Tris-Glycine SDS)
• Residue of the culture C2 1.1 and C2 1.2 (stored in 50 ml Falcon at -20°C)
• Protease inhibition PMSF at 100 mM
• Tris at 1 M
• NaCl at 5 M
• Laemli SDS buffer 2X
• Imidazole at 1.5 M
Method
Each sample has to be diluted with Laemmli 2X.
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
2. Follow the deposit table :
| L1 | L2 | L3 | L4 | L5 | L6 | L7 | L8 | L9 | L10 |
|---|---|---|---|---|---|---|---|---|---|
| 20 μl protein ladder | Ø | sample 19 | sample 21 | sample 23 | sample 25 | sample 27 | sample 29 | sample 31 | sample 33 |
Aim: Check if our fusion protein is efficient for silification.
Protocol: follow in this link
Materials
• Biochemical equipment : pipettes, cones, Eppendorf tubes, …
• TEOS (Tetraethyl Orthosilicate, Sigma)
• HCl 1 M
• Ulltrospec 3000 pro
• Fractions of fusion protein after FPLC purification
• Distilled water
• Buffer B
• 50 ml Falcons
Method
1.For fractions 27 to 31 :
1.a In a 50 ml Falcon, put 10 μl of our sample of protein in 10 ml of TEOS.
1.b In another 50 ml Falcon, put 10 μl of our sample of protein and 10 ml of water
2.For the other fractions :
2.a Prepare a solution of (Si(CH3O)4+HCl) with 55.8 μl of TEOS and 1 ml of HCl at 1 mM
2.b In a 50 ml Falcon, put 50 μl of our purified protein with the solution of (Si(CH3O)4+HCl) and let it stand for 4 minutes at room temperature
2.c Add 9 ml of buffer B
Results
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.
Hypothesis: for the concentration of our protein, 1 mg⁄ml correspond to 100 mM
The protein concentration was measured by its absorbance at 280 nm: A = 0.10400
We assumed to have an extinction coefficient of 1 OD280 nm= 1 mg⁄ml protein
Thus our [protein] = 0.104 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.
Aim: Make a preculture of BL21DE3 to increase the amount of bacteria available for innoculation later.
Protocol: follow in this link
Materials
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• carbenicillin at 50 mg⁄ml
• LB
• C2 1.2 colony
• 50 ml Erlenmeyer
• Shaking incubator at 37 °C
Method
1. In two Erlenmeyers of 50 ml, put 25 ml of LB and 25 μl of carbenicillin
2. Add in each Erlenmeyer one colony of C2 1.2 from the plate stored at 4°C
3. Put the two Erlenmeyers in the shaking incubator at 37°C and 150 rpm overnight.
