Difference between revisions of "Team:Pasteur Paris/Microbiology week7"

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:

*Table 55*
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

3. Add iPTG to have 1 mM concentration : C2 1.1 has a volume of 21 ml so the dilution factor is 500 from a stock of 0.5 M IPTG, so we added 42 μl of IPTG
4. After the last measurement of C2 1.1, the 1 ml sample is put in a 1.5 ml Eppendorf and centrifuged during 3 minutes at 7000 g. Then, throw away the supernatant; the pellet is stored with the label « before iPTG » at -20°C.
5. Measure the OD₆₀₀ :

*Table 56*
Colonies	OD₆₀₀
C2 1.1	0.928
C1 1.2	0.827

6. After the last measurement of C2 1.1, the sample is put in a 1,5 ml Eppendorf, centrifuge 3 minutes at 7000 g and once the supernatant has been thrown away, stored at -20°C called “C2 1.1 after iPTG”
7. The whole culture of C2 1.1 is centrifuged in a 50 ml Falcon during 15 minutes at 4500g. The supernatant is thrown away and the Falcon is stored at -20°C.
8. For the second culture C2 1.2 we decided to induce it later and we added iPTG to reach 0.3 mM.
9. The last measurement is pelleted in a 1.5 ml Eppendorf, the supernatant is thrown away and the Eppendorf is stored at -20°C
10. Our culture contains 19 ml so we add 11 &956;l of iPTG to have the right concentration
11. The Erlenmeyer is then put in the shaking incubator overnight at 37°C and 150 RPM

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 OD_{600 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 OD_{600 nm} of 10 :

*Table 57*
	OD_{600 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

2. Let lysate stand for 5 minutes at room temperature
3. Centrifuge for 10 minutes at 16000 g
4. In a clean 1.5 ml Eppendorf, add 10 μl of lysis product and 10 μl of laemmli 2X ⚠ Usually, the pellet and the supernatant are put in two different Eppendorfs, but our lysis buffer was too powerful and did not allow us to separate the two phases.
5. Put the Eppendorf 6 minutes at 93°C to denature the protein.

Aim: Check if our protein has been produced (weight around 30 kDa).

Method
Follow the deposit table :

*Table 58*
L1	L2	L3	L4	L5	L6	L7	L8	L9
Ladder	Ø	1.1(-)	Ø	1.1(+)	Ø	1.2(-)	Ø	1.2(+)

⚠ We follow exactly the same steps except we dilute the Bleu de Coomassie at 1⁄5 (7.5 ml of Bleu de Coomasie in 37.5 ml of final volume). We allow the staining to proceed for only 20 minutes.

Results
We did not observe at 30 kDa band for the induced cultures.
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.
We hypothesize that our protein is a fusion of the two proteins coded by the twinned insert.
We will send our recombinant plasmid to sequencing to check the ligation.
We will purify our protein on a Nickel column to test its efficiency in biosilification later.

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 Abs_{280 nm}
15. Collect 1 ml Fraction 15. All the fraction tubes are stored at 4°C

Results

Figure 3 Perform a SDS-PAGE separation of the fractions collected, as well as supernatant of crude lysate and pellet.
The protein is in the supernatant and not in the pellet.
After the purification, we noticed that some samples contain a protein that was hang onto the Nickel column.
We decided to do an SDS-PAGE gel to analyse the size of this protein

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 :

*Table 59*
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

3. Migration at 130 V for 45 minutes
4. Do 3 washes of 5 min with distilled water and then 50 minutes of coloration with Coomasie blue diluted 1⁄5

Results
A band located at 70 kDa appears for the sample 29

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 OD_{280 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.

@@ Line 285: / Line 285: @@
 . Measure the concentration of the cultures at several times  by absorbance at 600 nm: <br/>
 <table>
-<caption align="bottom" align="center">Table 1</caption>
+<caption align="bottom" align="center"><i><p> <U>Table 55</U></p></i></caption>
     <thead>
        <tr>
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 . Measure the OD<sub>600</sub> : <br/>
 <table>
-<caption align="bottom" align="center">Table 2</caption>
+<caption align="bottom" align="center"><i><p> <U>Table 56</U></p></i></caption>
     <thead>
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 . Dilution to reach an OD<sub>600 nm</sub> of 10 : <br/>
 <table>
-<caption align="bottom" align="center">Table 3</caption>
+<caption align="bottom" align="center"><i><p> <U>Table 57</U></p></i></caption>
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 Follow the deposit table : <br/>
 <table>
-<caption align="bottom" align="center">Table 4</caption>
+<caption align="bottom" align="center"><i><p> <U>Table 58</U></p></i></caption>
     <thead>
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 . Follow the deposit table :
 <table>
-<caption align="bottom" align="center">Table 5</caption>
+<caption align="bottom" align="center"><i><p> <U>Table 59</U></p></i></caption>
     <thead>
        <tr>

83. Checking of the gel

84. Culture of BL21DE3

July 19, 2016:

85. Continuation of the BL21DE3 cultures

86. Lysis of bacteria

87. Protein gel

July 20, 2016:

88. Purification on nickel columns

July 21, 2016:

89. SDS-PAGE gel

90. Silification tests

July 24, 2016:

91. Preculture of BL21DE3

Difference between revisions of "Team:Pasteur Paris/Microbiology week7"

Latest revision as of 00:53, 20 October 2016

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Week 7

July 18, 2016: