Team:Pasteur Paris/essai

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
• PSB1C3 plasmid (from shipped BioBrick-competent cells testing kit), chloramphenicol resistance
• pET43.1a (GE health care), ampicillin resistance (or carbenicillin)
• 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.
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.

Aim: To start a culture for Miniprep. 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
• 25 ml flasks
• Carbenicillin 50 mg/ml
• Chloramphenicol 34 mg/ml
• LB medium

Method:
1. One colony is picked from the plates and shaken in 25 ml of LB supplemented with Carbenicillin or Chloramphenicol at 50 g/ml or 34 g/ml respectively.
2. The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.

Aim: To perform a midiprep to isolate plasmid DNA of pSB1C3 and pET43.1a.
The amplification method to increase the amount of plasmid is called Mini or Midiprep.

Protocol: follow in this link

What we did in the lab:
Materials: • 50 ml Falcon tube
• Shaking incubator (INFORS HT)
• Carbenicillin 50 mg/ml
• Swing bucket centrifuge (JOUAN GR41)
• QIAGEN Midiprep kit 2016 (QiaFilter, Cat No.ID: 28704)

Method:
The protocol in step 1 ask for spinning at 6000g but we can only achieve 3500 g so we used 3500 g for 20 minutes. We will follow most of the protocol of QIAGEN Midiprep 2016 except for a few modifications, which we describe, therefore, below.
1. Use culture from overnight (17 hr) step on June 7, 2016
2. Pour culture in 50 ml Falcon nd centrifuge (15 min, 3500g, 4°C)
3. Discard the supernatant (in biological waste) and add 4 ml of Buffer P1 (stored on ice) to the pellet
4. Add 4 ml of Buffer P2 (for cell lysis) and mix by inverting the Falcon a few times. Wait 5 min at 22°C (room temperature: RT, EU). Note: The color of the solution will change to blue.
5. Prepare syringes with their cap and the reservoir (on 50 ml Falcon)
6. Add 4 ml of Buffer P3 (for neutralization) to the Falcon and mix by inverting the tube a few times. Note: The color of the solution changes to white.
7. Pour the content of the Falcon in the syringes and let it sit for 10 min. In the meanwhile, equilibrate the provided columns with 4 ml of OBT (equilibration buffer)
8. Transfer the contents from the syringe to the column and wash with 2 X 10 ml of QC buffer
9. Prepare 10 tubes of 2 ml to aliquot pET43.1 and pSB1C3.
Because we have only bench microfuges, we need to dispense our volume in smaller fractions.
10. Elution of DNA with 5 ml of QF and aliquot in 2 ml tubes
11. Centrifuge (30 min, 15000g, room temperature)
12. Add 3.5 ml of isopropanol, mix to precipitate the DNA
13. Centrifuge (30 min, 15 000g, at RT)
14. Remove isopropanol with pipet without taking DNA and place into chemical waste container
15. Add 1 ml of 70% ethanol, centrifuge again (15 min, 15 000g, RT) and let air dry
16. Resuspend in 50 L of Tris 10 mM pH 8.0, EDTA, 1 mM (TE) and store at -20°C

Aim: To linearize the different plasmids with appropriate enzymes.
We perform restriction enzyme digestion in order to recover linear backbones of the plasmids. We choose appropriate restriction sites based on the host plasmids.

Protocol: follow in this link

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

Method:
Measure the quantity of plasmid using a spectrophotometer (utrospec 3100 pro, Pharmacia GE health care)
Results:

λ= 260 nm	pET43.1 a(+)	pSB1C3
A₂₆₀	0.008	0.026
A₂₈₀	0.001	0.014
A₂₆₀/A₂₈₀	4	1.85
C diluted	0.4 ng/µl	1.9 ng/µl
C final	26.7 ng/µl	86.7 ng/µl

Table 1

The measured concentration after dilution were too low. We eventually switched to a Nanodrop (Thermofisher) because the plastic uvette gave too much background.

Method for digestion by Restriction enzymes
1. Mix all the reagents and let digest during 2 hr at 37°C
Big volumes must be added first!
• pET43.1 a at 87.7 ng/ will be digested by BamHI and Hind III (NEB)
• pSB1C3 at 26.7 ng/ will be digested by SpeI and XbaI (NEB)
2. We began the digestion 17h20. Here we digest 400 ng of DNA. We doubled Hind III volumes because this enzyme has only 50% of efficiency in Custmart (NEB) buffer.

	pSB1C3 (26.7 ng/µl)	pET43.1 a (86.7 ng/µl)
Vol SpeI	1 µl	0 µl
Vol XbaI	1 µl	0 µl
Vol HindIII	0 µl	2 µl
Vol BamHI	0 µl	1 µl
Vol H2O	28 µl	37.4 µl
Vol buffer 10X (Cutsmart)	5 µl	5 µl
Vol TOTAL	50 µl	50 µl

Table 2 3. Store at -20°C