Team:Pasteur Paris/Microbiology week14

Aim: Produce our protein in BL21(DE3) competent cells, the production is induced with IPTG once it reaches an OD_{600 nm} of 0.7.

Protocol: follow in this link

What we did in the lab:
Materials:
• Two 2 l erlenmeyers
• LB (Luria broth)
• IPTG (0.1 M)
• Precultures in 25 ml erlenmeyers
• UV spectrophotometer (Ultrospec 3100)
• Shaking incubator (INFORS HT)
• Centrifuge
• Buffer A (Tris-Cl 50 mM pH 7.4, NaCl 150 mM)

Method:
1. Put 1 l of LB in each erlenmeyer and make them warm with the shaking incubator at 37°C and 150 rpm.
2. Once warmed, add 12.5 ml of preculture in each erlenmeyer.
3. Let grow in the shaking incubator.
4. Measure the absorbance with the UV spectrophotometer every 30 minutes.

*Table 141 : Optical Density*
Time	C2 (1)	C2 (2)
10:30 _AM	0	0
01:55 _PM	0.438	/
2:25 _PM	0.602	0.429
2:45 _PM	0.679	0.600

5. Add IPTG to reach a concentration of 0.1 mM. (3:00 _PM)
6. The last measure before induction is pelleted 3 min at 8000g and stored at -20°C.
  7. Let induce overnight in the shaking incubator at 15°C at 150 rpm
8. The day after, measure the OD_{600 nm} and store the measure pelleted at -20°C.
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.

Aim: Purifying the protein C2 produced by BL21(DE3) using a Fast Purification Liquid Chromatography. We use the culture induced the previous day.

Protocol: follow in this link

What we did in the lab:
Materials:
• Fast Purification Liquid Chromatography
• Chaotropic reagent (Guanidinium HCL 6 M)
• EDTA 0,1 M
• PMSF (100 mM)
• Ni²⁺ solution (100 mM)
• Centrifuge
• Buffer A (Tris 50 mM pH 7.4, NaCl 150 mM)
• Buffer B (Tris 50 mM pH 7.4, NaCl 150 mM, Imidazole 250 mM)

Method:
1. Melt the pellet of bacteria C2 (from 1 l culture) and resuspend it with 10 ml of buffer A. We add 25 ml of pellet we complete until 40 ml with buffer A.
2. Add 40 µl of PMSF to avoid protein denaturation.
3. Put the column on the FPLC, clean the FPLC with water and then fill it with buffer A.
4. Sonicate the sample three times one minute at 60%, wait 90 seconds between each sonication.
5. Centrifuge 25 min at 16000g (rotor Beckman JA 25.50)
6. Inject your sample in the FPLC
7. Get back several samples:
→ C: Crude extract : before centrigugation
→ P: Pellet
→ SN: Supernatant
→ F: Flow through (unfixed proteins)
→ W: Wash 5% of buffer B
→ Fractions (depending on the gradient)

Aim: Get the size of the protein purified thanks to FPLC in order to know if it is our protein.

Material: • SDS-PAGE chamber
• SDS-PAGE gel 4-15% gradient (BIORAD)
• Protein migration buffer TGS 20X
• Protein ladder PAGE Ruler (Thermofisher)
• Laemmli 2X
• Coomassie Blue
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)

Method:
1. In nine 1.5 ml Eppendorf, put 20 µl of a sample and 20 µl of Laemmli 2X.
2. Let denaturate the proteins 5 minutes at 95°C.
3. Place the gel into the chamber and fill it with migration buffer.
4. Follow the next deposit table:

Fraction 13 | Fraction 15 | Fraction 17 | Fraction 19 | Fraction 21 | Fraction 23 | Fraction 25 | Fraction 27 | Protein gene ruler (8 µl)

5. Launch the migration at 130V.
6. Wash the gel three times with distilled water during 5 minutes.
7. Color the gel with Coomassie Blue diluted 1/5 during 30 minutes.
8. Wash with distilled water for 5 minutes then let wash 15 minutes.

Aim: To start a culture for Miniprep of insert A1 / A2 / B1 / B2 / C1 / C2 / D1 / D2 / E1 / E2.
In order to obtain a large amount of plasmid, we need to grow the bacteria overnight.

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)
• 15 ml Falcons
• Carbenicillin 50 mg/ml
• LB medium

Method:
1. One colony is picked from the plates and shaken in 5.0 ml of LB supplemented with Carbenicillin at 50 µg/ml.
2. The flask is placed in a shaking incubator at 37°C and 150 rpm overnight.

Aim: To perform a Midiprep to isolate plasmid DNA of pET43.1(a+) with the inserts A1 / A2 / B1 / B2 / C1 / C2 / D1 / D2 / E1 / E2 from cultures made on the 8/09.
The amplification method to increase the amount of plasmid is called Midiprep.
Protocol: follow in this link

What we did in the lab:
Materials:
• 50 ml Falcon tube
• Shaking incubator (INFORS HT)
• Swing bucket centrifuge (JOUAN GR41)
• QIAGEN Miniprep kit
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)

Method:
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.
2. Follow QIAGEN kit steps. Final volume: 100 µl

Aim: Measure the quantity of plasmid using a Nanodrop (Thermofisher)

What we did in the lab:
Materials:
• Nanodrop (Thermofisher)
• Elution buffer from QIAGEN kit
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)

Method:
1. Analyze absorbance at 260nm.
2. Clean the Nanodrop with water.
3. Make the blank with 1 µl of elution buffer.
4. Put 1 µl of your sample on the Nanodrop.
5. Make the measure and clean the Nanodrop between each measure.

Results:

*Table 142 : Absorbance*
Absorbance at 260nm	A₂₆₀	A₂₈₀	A_260/280	Concentration (ng/µl)
A1	0.645	0.345	1.87	32.3
A1’ _{(diluted 1/2)}	1.314	0.704	1.86	65.7
A2_{(diluted 1/4)}	1.404	0.752	1.87	70.2
A2’_{(diluted 1/4)}	0.494	0.269	1.87	24.7
C1_{(diluted 1/4)}	0.305	0.165	1.85	15.2
C1’_{(diluted 1/4)}	0.812	0.445	1.83	40.6
C2_{(diluted 1/4)}	0.499	0.272	1.83	24.9
C2’_{(diluted 1/4)}	0.757	0.402	1.88	37.8
D1_{(diluted 1/4)}	0.441	0.233	1.89	22.1
D1’_{(diluted 1/4)})	0.725	0.396	1.83	36.3
D2_{(diluted 1/4)})	0.264	0.141	1.87	13.2
D2’_{(diluted 1/4)})	0.418	0.216	1.94	20.9
E1_{(diluted 1/4)})	0.487	0.266	1.83	24.3
E1’_{(diluted 1/4)})	0.478	0.247	1.94	24.0
E2_{(diluted 1/4)})	1.364	0.742	1.84	68.2
E2’_{(diluted 1/4)})	1.041	0.562	1.85	52.1

Team:Pasteur Paris/Microbiology week14

click on the weeks

Microbiology Notebook

Week 14

September 6, 2016:

236. Growth of bacteria

September 7, 2016:

237. Purification of the protein

September 8, 2016:

238. Protein gel on SDS-PAGE

239. Harvest the culture with Miniprep

September 9, 2016:

240. Extraction of plasmid DNA

241. Measure the amount of DNA extracted from the miniprep