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

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Week 7

July 18, 2016:

July 19, 2016:

85. Continuation of the BL21DE3 cultures

July 20, 2016:

88. Purification on nickel columns

July 21, 2016:

July 24, 2016:

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: 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: 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

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