Team:Pasteur Paris/essai

Aim: To perform a gel extraction to isolate insert DNA purified from its plasmid thanks to the migration. We use the gel made before with inserts B2/E1/E2 but we only extract B2 bands.

What we did in the lab:
Materials:
• scalpel
• 2 ml eppendorfs
• balance
• UV table
• microbiology equipment
• QIAGEN Gel Extraction Kit

Method:
Be aware of the risks! UV light burns the eyes and skin so make sure you have the right protection
Follow QIAGEN Kit steps according to the next tables for the volumes of QG buffer.

Results:

lambda=260nm	Concentration (ng/µL)
C₁	3.5
C₂	2.9
C₃	3.4
C₄	4.2
C₅	4.1
C₆	15.1
C₇	5.9
C₈	4.9
C₉	4.0
C₁₀	4.9
C₁₁	4.3
C₁₂	4.1
C₁₃	7.2
C₁₄	4.9
C₁₅	4.7
C₁₆	8.5
C₁₇	4.4
C₁₈	3.6
C₁₉	5.2

Concentrations

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 (Follow this link)

Method:
Analyze absorbance at 260nm
Clean the Nanodrop with water
Make the blank with 1 µL of elution buffer
Put 1 µL of your sample on the Nanodrop
Make the measure and clean the Nanodrop between each measure

Results:

lambda=260nm	Concentration (ng/µL)
C₁	3.5
C₂	2.9
C₃	3.4
C₄	4.2
C₅	4.1
C₆	15.1
C₇	5.9
C₈	4.9
C₉	4.0
C₁₀	4.9
C₁₁	4.3
C₁₂	4.1
C₁₃	7.2
C₁₄	4.9
C₁₅	4.7
C₁₆	8.5
C₁₇	4.4
C₁₈	3.6
C₁₉	5.2

Concentrations

Aim:To increase the amount of plasmid by transformation in competent cells.
The amount of plasmid supplied is insufficient to perform all our future experiments. Therefore, we need to amplify the amount of plasmids.

Protocol: follow in this link

What we did in the lab:
Materials:
• subcloning competent cells TOP10
• pET43.1a (GE health care), ampicillin resistance (or carbenicillin) ligated with the inserts
• SOC (Super optimal Broth) media
• LB (lysogeny Luria broth) Agar plates containing 50 mg/ml carbenicillin or 34 mg/ml chloramphenicol
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etcfollow this link)

Method:
1.Thaw cells from -80°C on ice, thaw plasmid at 37°C and store on ice, aliquot cells for 50 µL/vial.
2. Add 50 pg plasmids to each 50 µL of competent cells vial and tap gently. Here, add 12.5µL of DNA.
3. Place on ice for 30 min. Meanwhile, warm LB agar plates from cold room in 37°C non shaking incubator.
4. Place cells in 42°C water bath for exactly 40 seconds and then place immediately on ice for at least 3 min.
5. Add 500 µL of SOC in each tube and place them to shaking incubator (incline tube for better shaking efficiency).
6. Grow for 40 minutes at 37°C in shaking incubator at 150 rpm.
7. Near Bunsen burner flame, add 45 µL of competent cells +135µL of SOC or 200 µL aliquots of culture and streak plate with sterile rake on LB-agar plates containing the appropriate antibiotic.
8. Place plates inverted in the static 37°C incubator overnight.

Results:
The transformation works we had a lot of colonies, we stored them at 4°C to stop their growth.

Aim: Purifying the protein produced by BL21(DE3) using a Fast Protein Liquid Chromatography.
We pool two pellets of bacteria produced in 1L of culture each.

What we did in the lab:
Materials:
• Fast Protein Liquid Chromatography
• Chaotropic reagent (Guanidinium 6M)
• EDTA 0,1M
• PMSF (100mM)
• Ni 2+ solution (100mM)
• Centrifuge (labo deshmukh)
• Buffer A (50mM Tris, 150mM of NaCl)
• Buffer B (50mM Tris, 150mM of NaCl, 250mM Imidazole, pH=7.4)

Method:
Melt the pellet of bacteria C2 (from 1L culture) and resuspend it with 10ml of buffer A. We had 25ml of pellet we complete until 40ml with buffer A.
Add 40 ul of PMSF
Put the column off the FPLC and wash it with 20 ml of milliQ water thanks to a fingerpit ans a syringue.
Add 20ml of chaotropic reagent to denaturate the proteins fixed to the column
Wash the column with 20ml of water
Add 10ml of EDTA to clean it from nickel
Wash with 20ml of water
Add 5ml of Ni solution to charge the column. The column turns green.
Wash with 20ml of water
Sonicate the sample three times one minute at 60%, wait 90 seconds between each sonication.
Finally, the sample is 40 ml, add 40 ul of PMSF to avoid protein denaturation.
Centrifuge 25 min at 16000g (rotor JA 25.50)
Inject your sample in the FPLC
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

What we did in the lab:
Materials:
• SDS-Page cuve
• SDS-Page gel (BIORAD)
• Protein migration buffer
• Protein ladder
• Laemmli 2X
• Coomassie Blue
• Microbiology equipment (Follow this link)

Method:
In 9 1.5ml eppendorf, put 20 µL of a sample and 20 µL of Laemmli 2X.
Let denaturate the proteins 5min at 95°C
Place the gel into the cuve and fill it with migration buffer
Follow the next deposit table:
• Protein ruler 8 µL
• Crude extract
• Supernatant
• Pellet
• Wash
• Fraction 11
• Fraction 13
• Fraction 15
• Fraction 18
• Fraction 19
• Fraction 20
• Fraction 21
4. Launch the migration at 130V.
5. Wash the gel three times with distilled water during 5min.
6. Color the gel with Coomassie Blue diluted 1/5 during 30min.
7. Wash with distilled water for 5min then let wash 15min.

Aim: To perform a Miniprep to isolate plasmid DNA of pET43.1a with the inserts A1/A2 and D1/D2 from cultures made on the 22/08.
The amplification method to increase the amount of plasmid is called Miniprep. Only 25 colonies grow.

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:
The protocol in step 1 ask for spinning at 6000g but we can only achieve 3500 g so we used 3500 g for 8 minutes. We will follow most of the protocol of QIAGEN Miniprep 2016 except for a few modifications, which we describe, therefore, below.
Follow QIAGEN kit steps

Aim:To get back our insert from the Miniprep with appropriate enzymes. We perform restriction enzyme digestion in order to recover our inserts.
We choose appropriate restriction sites based on the host plasmid.
A1(7 tubes) / A2 (6 tubes) / D1(6 tubes) / D2(6 tubes)

Protocol: follow in this link

What we did in the lab:
Materials:
• Restriction enzymes: XbaI, HindIII (New England Biolabs, NEB)
• Restriction enzyme buffers
• 37°C water bath
• UV spectrophotometer

Method:
Mix all the reagents and let digest during 2 hr at 37°C
Big volumes must be added first! Make a global mix to be more accurate as we have 25 tubes.
Beginning of digestion 12:00AM.

Reactants	Each sample	Global mix
Vol_DNA	25 µL	0 µL
Vol_XbaI	1 µL	25 µL
Vol_HindIII	1 µL	25 µL
Vol_H2O	0 µL	0 µL
Vol_{Buffer 2.1}	3 µL	75 µL
Vol_total	30 µL	125 µL

Volumes

2. Incubate 10 min at 65°C to inactivate the enzymes.
3. Store at -20°C

Aim: This step check the digestion efficiency of A1(7 tubes) / A2 (6 tubes) / D1(6 tubes) / D2(6 tubes).
Moreover, the inserts will be purified during this step because they will be extracted from the gel.

Protocol: follow in this link

What we did in the lab:
Materials:
• Electrophoresis cuve
• TAE 1X
• Gene ruler (Thermoscientific 1kb plus)
• Loading dye
• Agarose
• UV table
• BET

Method:
Each well can contain 40ul so we made a big gels with 20x2 lines. Each sample will contain 36ul as we add 6ul of loading dye. Deposit table (/// means EMPTY to make the cut easier)

Gel 1 Line 1 :
Ladder /// A1(0) / A1(1) / A1(3) / A1(5) / A1(6) / A1(7) / A1(8) /// A2(1) / A2(2) / A2(5) / A2(6) / A2(7) / A2(8)

Gel 1 Line 2 :
Ladder /// D1(1) / D1(3) / D1(4) / D1(5) / D1(8) / D1(9) /// D2(2) / D2(4) / D2(5) / D2(7) / D2(8) / B2(10)

Aim: To start a culture for Miniprep of insert E1 and E2 from cells transformed on the 22/08.
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 equipement
• 1 ml eppendorfs
• Carbenicillin 50 mg/ml
• LB medium

Method:
One colony is picked from the plates and shaken in 1.0 ml of LB supplemented with Carbenicillin at 50 μg/ml. 20 colonies are taken from each insert.
The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.

Aim: To recircle the dephosphorylated plasmid pET43.1a with the insert before the transformation in competent cells.

Protocol: follow in this link

What we did in the lab:
Materials:
• Ligation enzymes: T4 ligase (New England Biolabs, NEB)
• Ligation buffer 10X
• 65°C heat table
• 100ng of pET43.1a plasmid (6.8ng/µL)
• 50ng of purified insert B2 (15.8ng/µL)

Method:
Mix all the reagents and let digest during 30 min at room temperature.
Big volumes must be added first!

Reactants	B2	pET43.1a
Vol_{plasmid DNA}	14.5 µL	14.5 µL
Vol_Insert	3.2 µL	0 µL
Vol_{ligation buffer}	3.7 µL	3.7 µL
Vol_H2O	14.5 µL	17.7 µL
Vol_{T4 ligase}	1 µL	1 µL
Vol_total	36.9 µL	36.9 µL

Volumes

2. Incubate 5 min at 65°C to inactivate the enzymes.
3. Store at -20°C

Aim: To increase the amount of plasmid by transformation in competent cells.
The amount of plasmid supplied is insufficient to perform all our future experiments. Therefore, we need to amplify the amount of plasmids.

Protocol: follow in this link

What we did in the lab:
Materials:
• subcloning competent cells TOP10
• pET43.1a (GE health care), ampicillin resistance (or carbenicillin) ligated with the inserts
• SOC (Super optimal Broth) media
• LB (lysogeny Luria broth) Agar plates containing 50 g/ml carbenicillin or 34 g/ml chloramphenicol
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etcfollow this link)

Method:
1.Thaw cells from -80°C on ice, thaw plasmid at 37°C and store on ice, aliquot cells for 50 µL/vial.
2. Add 50 pg plasmids to each 50 µL of competent cells vial and tap gently. Here, add 12.5ul of DNA.
3. Place on ice for 30 min. Meanwhile, warm LB agar plates from cold room in 37°C non shaking incubator.
4. Place cells in 42°C water bath for exactly 40 seconds and then place immediately on ice for at least 3 min.
5. Add 500 µL of SOC in each tube and place them to shaking incubator (incline tube for better shaking efficiency).
6. Grow for 40 minutes at 37°C in shaking incubator at 150 rpm.
7. Near Bunsen burner flame, add 45 µL of competent cells +135µL of SOC or 200 µL aliquots of culture and streak plate with sterile rake on LB-agar plates containing the appropriate antibiotic.
8. Place plates inverted in the static 37°C incubator overnight.

Results:
The transformation works we had a lot of colonies, we stored them at 4°C to stop their growth.

Aim: We realize that our column was made of sepharose, a material similar to cellulose and our protein might be retained.
We decided to clean the column with urea.

Protocol: follow in this link

What we did in the lab:
Materials:
• Urea (6M)
• Buffer A (50mM Tris, 150mM of NaCl)
• 50 ml Falcon flasks
• Dialysis MNCO membrane
• Columns used for the purification
• Syringues
• Microbiology equipment (Follow this link)

Method:
1. Inject 5ml of urea solution with a syringue in the column used to get back Prep1 . Get back the flow throught in a Falcon and let denaturate for 5min.
2. Inject 5ml of urea in the column and get back the flow throught in the Falcon. 10ml of products
3. Dialyse the content of the Falcon with the membrane MNCO, 3.500, soaking in 500ml of buffer A overnight, 4°C
4. Do the same protocol with Prep 2
5. Inject 5ml of buffer A in each column to clean them of urea
6. Store at RT samples before dialysis and after dialysis

Aim: To get back our insert from the Miniprep with appropriate enzymes.
We perform restriction enzyme digestion in order to recover our inserts. We choose appropriate restriction sites based on the host plasmid.
C1(10 tubes)

Protocol: follow in this link

What we did in the lab:
Materials:
• Restriction enzymes: XbaI, HindIII (New England Biolabs, NEB)
• Restriction enzyme buffers
• 37°C water bath
• 65°C heating table

Method:
Mix all the reagents and let digest during 2 hr at 37°C
Big volumes must be added first! Make a global mix to be more accurate as we have 25 tubes.
Beginning of digestion 1:07PM.

Reactants	Each sample	Global mix
Vol_DNA	20 µL	0 µL
Vol_XbaI	1 µL	10 µL
Vol_HindIII	1 µL	10 µL
Vol_H2O	0.5 µL	5 µL
Vol_{Buffer 2.1}	2.5 µL	25 µL
Vol_total	25 µL	50 µL

Volumes

2. Incubate 10 min at 65°C to inactivate the enzymes.
3. Store at -20°C

Aim: This step check the digestion efficiency of C1(10 tubes).
Moreover, this step is done to check the digestion efficiency not to purify the inserts.

Protocol: follow in this link

What we did in the lab:
Materials:
• Electrophoresis cuve
• TAE 1X
• Gene ruler (Thermoscientific 1kb plus)
• Loading dye
• Agarose
• UV table
• BET
Method:
Make a small gel with 12 wells. Each sample will contain 6ul as we add 1ul of loading dye. Deposit table (/// means EMPTY to make the cut easier)

Ladder /// C1(1) / C1(2) / C1(3) / C1(4) / C1(5) / C1(6) / C1(7) / C1(8) / C1(9) / C1(10)

Results:
the digestion works since we can clearly notice two distincts bands in each well. We will redo this gel to purify the inserts with all the DNA we have (refer on the 25/08)

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

What we did in the lab:
Materials:
• SDS-Page cuve
• SDS-Page gel (BIORAD)
• Protein migration buffer
• Protein ladder
• Laemmli 2X
• Coomassie Blue
• Microbiology equipment (Follow this link)
Method:
In 9 1.5mL eppendorf, put 10 µL of a sample and 10 µL of Laemmli 2X.
Let denaturate the proteins 5min at 95°C
Place the gel into the cuve and fill it with migration buffer
Follow the next deposit table:
• Protein ruler 8 µL
• ///
• Prep 1 non dialysed
• Prep 2 non dialysed
•///
• Prep 1 dialysed
• Prep 2 dialysed
•///
• Buffer A used for the dialysis
• ///
• column wash
•///
4. Launch the migration at 130V. Start of the migration 4:10PM
5. Wash the gel three times with distilled water during 5min.
6. Color the gel with Coomassie Blue diluted 1/5 during 30min.
7. Wash with distilled water for 5min then let wash 15min.

Aim: To perform a Miniprep to isolate plasmid DNA of pET43.1a with the inserts E1/E2 from cultures made on the 23/08.
The amplification method to increase the amount of plasmid is called Miniprep.
E1 (19 tubes, one did not grow) / E2 (20 tubes)

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:
The protocol in step 1 ask for spinning at 6000g but we can only achieve 3500 g so we used 3500 g for 8 minutes. We will follow most of the protocol of QIAGEN Miniprep 2016 except for a few modifications, which we describe, therefore, below.

Follow QIAGEN kit steps

Aim: To get back our insert from the Miniprep with appropriate enzymes.
We perform restriction enzyme digestion in order to recover our inserts. We choose appropriate restriction sites based on the host plasmid.
E1 (19 tubes, one did not grow) / E2 (20 tubes)

Protocol: follow in this link

What we did in the lab:
Materials:
• Restriction enzyme: HindIII (New England Biolabs, NEB)
• Restriction enzyme + buffer : XbaI Remix (New England Biolabs, NEB)
• 37°C water bath
• 65°C heating table

Method:
Mix all the reagents and let digest during 2 hr at 37°C
Big volumes must be added first! Make a global mix to be more accurate as we have 39 tubes.

Reactants	Each sample	Global mix
Vol_DNA	25 µL	0 µL
Vol_{XbaI Remix}	5 µL	200 µL
Vol_HindIII	4 µL	160 µL
Vol_H2O	16 µL	640 µL
Vol_total	50 µL	1000 µL

Volumes

2. Incubate 10 min at 65°C to inactivate the enzymes.
3. Store at -20°C

Aim: Produce our protein in BL21(DE3) competent cells, the production is induced with IPTG once it reeaches an optical density of 0.7.

Protocol: follow in this link

What we did in the lab:
Materials:
• 4 2L erlenmeyers
• LB (lysogeny Luria broth)
• IPTG (0.1M)
• Precultures in 25ml erlenmeyers
• UV spectrophotometer (Ultrospec 3100)
• Shaking incubator (INFORS HT)
• Centrifuge
• Buffer A (50mM Tris, 150mM of NaCl)

Method:
Put 1L of LB in each erlenmeyer and make them warm with the shaking incubator at 37°C and 150RPM
Once warmed, add 5ml of preculture in each erlenmeyer
Let grow in the shaking incubator. Start of growth at 11:10AM.
Measure the absorbance with the UV spectrophotometer every 30min

Time	C2(1)	C2(2)	C2(3)	C2(4)
2:22_PM	0.113	0.132	0.203	0.143
2:55_PM	0.303	0.339	0.421	0.328
3:38_PM	0.476	0.509	0.593	0.494
3:52_PM	0.614	0.659	0.683	0.609

Absorbance

5. Add IPTG to reach a concentration of 0.1mM.
6. The last measure before induction is pelleted 3min at 8000g and stored at -20°C.
 7. Let induce overnight in the shaking incubator
8. The day after, measure the OD and store the measure pelleted at -20°C. We measure 1.195
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: This step check the digestion efficiency of E1 and E2. E1 (19 tubes, one did not grow) / E2 (20 tubes)
Moreover, the inserts will be purified during this step because they will be extracted from the gel.

Protocol: follow in this link

What we did in the lab:
Materials:
• Electrophoresis cuve
• TAE 1X
• Gene ruler (Thermoscientific 1kb plus)
• Loading dye
• Agarose
• UV table
• BET

Method:
Make two big gels with 2x20 wells and a small gel with 12 wells. Each sample will contain 60 µL as we add 10 µL of loading dye to the 50 µL of DNA.
Deposit table (/// means EMPTY to make the cut easier)

Gel 1 Line 1 :
Ladder /// E1(10) / E1(1) / E1(1) / E1(2) / E1(2) / E1(3) / E1(3) / E1(4) / E1(4) / E1(5) / E1(5) / E1(6) / E1(6) / E1(7) / E1(7) / E1(8) / E1(8) / E1(9) / E1(9)

Gel 1 Line 2 :
Ladder /// E1(10) / E1(11) / E1(11) / E1(12) / E1(12) / E1(13) / E1(13) / E1(14) / E1(14) / E1(15) / E1(15) / E1(16) / E1(16) / E1(17) / E1(17) / E1(18) / E1(18) / E1(19) / E1(19)

Gel 2 Line 1 :
Ladder /// E2(1) / E2(1) / E2(2) / E2(2) / E2(3) / E2(3) / E2(4) / E2(4) / E2(5) / E2(5) / E2(6) / E2(6) / E2(7) / E2(7) / E2(8) / E2(8) / E2(9) / E2(9)

Gel 2 Line 2 :
Ladder /// E2(10) / E2(10) / E2(11) / E2(11) / E2(12) / E2(12) / E2(13) / E2(13) / E2(14) / E2(14) / E2(15) / E2(15) / E2(16) / E2(16) / E2(17) / E2(17) / E2(18) / E2(18)

Small gel:
Ladder /// E2(19) / E2(19) / E2(20) / E2(20) / E1(19) / E1(19)

Aim: This step check the ability of our protein to bind cellulose. Moreover, this step will confirm the good design of the protein.

Protocol: follow in this link

What we did in the lab:
Materials:
• Cellulose Avicell (Sigma-Aldrich)
• BSA Protein
• Rocker
• Centrifuge
• Laemmli 2X
• Buffer A (50mM Tris, 150mM of NaCl)
• Heating table
• SDS-Page gel and cuve
• Protein ruler
• Microbiology equipment

Method:
Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)
In a 1.5mL eppendorf, put 1mL of protein solution (previously purified thanks to FPLC), 100 µL of BSA solution and 10mg of Avicell.
Let rocking 1h at room temperature
Centrifuge 2min at 13.000 rpm
Take 1mL of the supernatent and store it in a 2mL eppendorf
Resuspend the pellet in 1mL of Buffer A
Vortex and let rocking during 2min at RT
Centrifuge 2min at 13.000rpm
Collect the supernatent and pool it with the first taken
Resuspend the pellet in 800 µL of Laemmli 2X
Denaturate the protein by heating them at 95°C during 5min
Centrifuge 2min at 13.000rpm
Depose 20 µL of the supernatent
For the other samples: mix 10 µL of Laemmli 2X with 10 µL of protein (BSA alone, Prep2 after dialysis). Denaturate them 5min at 95°C

Deposit table on the gel:
• Protein ruler
•///
• Pellet
•///
 • Supernatant
•///
• Prep2
•///
 • BSA

Start at 10:15AM

15. Wash the gel three times with distilled water during 5min.
16. Color the gel with Coomassie Blue diluted 1/5 during 30min.
17. Wash with distilled water for 5min then let wash 15min.

Aim: This step check the ability of our protein to catalyse silification.
Moreover, this step will confirm the good design of the protein.

What we did in the lab:
Materials:
• HCl
• TEOS
• 15mL Falcons

Method:
Make one test with two control tubes
1mL of HCl + 1mL of TEOS, 4min at RT then add 100 µL of protein solution Prep 2 after dyalisis and 8mL of Buffer A
1mL of HCl + 8mL of Buffer A , 4min at RT then add 100 µL of the protein solution (no silicic acid is produced)
1mL of HCl + 1mL of TEOS, 4min at RT then add 8mL of Buffer A (no protein control)

Start at 8:45PM

Aim: This step check the ability of our protein to bind cellulose. Moreover, this step will confirm the good design of the protein.

What we did in the lab:
Materials:
• Cellulose Avicell (Sigma-Aldrich)
• Cellulose Sigmacell (Sigma-Aldrich)
• Carboxymethylcellulose (Sigma-Aldrich)
• BSA Protein
• Rocker
• Centrifuge
• Laemmli 2X
• Buffer A (50mM Tris, 150mM of NaCl)
• Heating table
• SDS-Page gel and cuve
• Protein ruler
• Microbiology equipment

Method:
This protocol is done three times with the different cellulose.
Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)
In a 1.5mL eppendorf, put 1mL of protein solution (previously purified thanks to FPLC), 100 µL of BSA solution and 10mg of Avicell.
Let rocking 1h at room temperature
Centrifuge 2min at 13.000 rpm
Take 1mL of the supernatent and store it in a 2mL eppendorf
Resuspend the pellet in 1mL of Buffer A
Vortex and let rocking during 2min at RT
Centrifuge 2min at 13.000rpm
Collect the supernatent and pool it with the first taken
Resuspend the pellet in 800 µL of Laemmli 2X
Denaturate the protein by heating them at 95°C during 5min
Centrifuge 2min at 13.000rpm
Depose 20 µL of the supernatent
For the other samples: mix 10 µL of Laemmli 2X with 10 µL of protein (BSA alone, Prep2 after dialysis). Denaturate them 5min at 95°C

Deposit table on the gel:
• Protein ruler
•///
• Pellet (BSA + protein + Sigmacell)
• Supernatant (BSA + protein + Sigmacell)
• Pellet (BSA + Sigmacell)
• Supernatant (BSA + Sigmacell)

• Pellet (BSA + protein + Avicell)
• Supernatant (BSA + protein + Avicell)

• Pellet (BSA + protein + CMC)
• Supernatant (BSA + protein + CMC)
• Pellet (BSA + CMC)
• Supernatant (BSA + CMC)

Start at 11:10AM at 130V. End at 12:20AM.
15. Wash the gel three times with distilled water during 5min.
16. Color the gel with Coomassie Blue diluted 1/5 during 30min.
17. Wash with distilled water for 5min then let wash 15min.

Aim: To start a culture for Miniprep of insert A1/A2 and D1/D2. 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 equipement
• 15 mL Falcons
• Carbenicillin 50 mg/ml
• LB medium

Method:
One colony is picked from the plates and shaken in 5.0 mL of LB supplemented with Carbenicillin at 50 µg/ml. 4 colonies are taken from each insert.
The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.

Aim: To perform a Miniprep to isolate plasmid DNA of pET43.1a with the inserts A1/A2 and D1/D2 from cultures made on the 25/08.
The amplification method to increase the amount of plasmid is called Miniprep.
Only 25 colonies grow.

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:
The protocol in step 1 ask for spinning at 6000g but we can only achieve 3500 g so we used 3500 g for 8 minutes. We will follow most of the protocol of QIAGEN Miniprep 2016 except for a few modifications, which we describe, therefore, below.

Colonies that grow: A1(1), A1(2), A1(3), A2(1), A2(2), A2(3), A2(4), D1(2), D2(1), D2(2), D2(3), D2(4),

Follow QIAGEN kit steps

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 (Follow this link)

Method:
Analyze absorbance at 260nm
Clean the Nanodrop with water
Make the blank with 1 µL of elution buffer
Put 1 µL of your sample on the Nanodrop
Make the measure and clean the Nanodrop between each measure

Results:

Absorbance at 260nm (diluted 1/10)	A260	A280	A260/280	Concentration (ng/µL )
A1(1)	0.393	0.206	1.91	19.6
A1(2)	0.460	0.245	1.87	23.0
A1(3)_{non diluted}	1.593	0.850	1.87	79.7
A2(1)	0.381	0.219	1.74	19.1
A2(2)	0.303	0.150	2.02	15.1
A2(3)_{non diluted}	0.211	0.111	2.0	11.1
A2(4)	0.280	0.158	1.78	14.0
D1(2)_{non diluted}	0.274	0.148	1.85	13.7
D2(1)	1.740	0.911	1.91	87.0
D2(2)	0.274	0.151	1.82	13.7
D2(3)_{non diluted}	0.214	0.128	1.67	10.7
D2(4)	0.393	0.256	1.55	19.6

Absorbance

Aim: To get back our insert from the Miniprep with appropriate enzymes.
We perform restriction enzyme digestion in order to recover our inserts. We choose appropriate restriction sites based on the host plasmid.
A1(3 tubes) / A2 (4 tubes)

Protocol: follow in this link

What we did in the lab:
Materials:
• Restriction enzymes: HindIII (New England Biolabs, NEB)
• Restriction enzyme + buffer : XbaI Remix (New England Biolabs, NEB)
• 37°C water bath
• UV spectrophotometer

Method:
Mix all the reagents and let digest during 2 hr at 37°C
Big volumes must be added first! Make a global mix to be more accurate as we have 25 tubes.

Beginning of digestion 12:15AM.

Reactants	Each sample	Global mix
Vol_DNA	25 µL	0 µL
Vol_{XbaI Remix}	5 µL	35 µL
Vol_HindIII	4 µL	28µL
Vol_H2O	16 µL	112 µL
Vol_total	50 µL	175 µL

Volumes

2. Incubate 10 min at 65°C to inactivate the enzymes.

Aim: This step check the digestion efficiency of A1(3 tubes) / A2 (4 tubes).
Moreover, the inserts will be purified during this step because they will be extracted from the gel.
We also depose inserts D1 and D2 undigested to check.

Protocol: follow in this link

What we did in the lab:
Materials:
• Electrophoresis cuve
• TAE 1X
• Gene ruler (Thermoscientific 1kb plus)
• Loading dye
• Agarose
• UV table
• BET

Method:
Each well can contain 40 µL so we made a big gel with 20x2 lines. Each sample will contain 36 µL as we add 6 µL of loading dye. Deposit table (/// means EMPTY to make the cut easier)

Gel 1 Line 1 :
Ladder /// A1(1) / A1(1) /// A1(2) / A1(2) /// A1(3) / A1(3) /// A2(1) / A2(1) ///A2(2) / A2(2) /// A2(3) / A2(3) /// ///

Gel 1 Line 2 :
Ladder /// D1(1) /// D1(2) /// D2(4) /// D2(1) /// D2(2) /// D2(3)

Results:
All the digestion have worked except A1(3) that seems to be contaminated. Moreover, D2(2) and D2(3) seems to have the insert so we will have to digest them.

Aim: To perform a gel extraction to isolate insert DNA purified from its plasmid thanks to the migration. We use the gel made before with inserts B2/E1/E2 but we only extract B2 bands.

Protocol: follow in this link

What we did in the lab:
Materials:
• scalpel
• 2 ml eppendorfs
• balance
• UV table
• microbiology equipment
• QIAGEN Gel Extraction Kit

Method:
Be aware of the risks! UV light burns the eyes and skin so make sure you have the right protection
Follow QIAGEN Kit steps according to the next tables for the volumes of QG buffer.

Insert	Mass of gel (mg)	Volume of QG Buffer (µL )
A1(1)	482	1446
A1(2)	491	1473
A2(1)	478	1434
A2(2)	395	1185
A2(3)	360	1080
A2(4)	472	1416

Masses

August 23, 2016:

August 24, 2016:

August 25, 2016:

August 26, 2016:

Team:Pasteur Paris/essai

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August 22, 2016: