Protocols
Strains used :
In this project we used two strains of E.Coli. MC1000 to express our banks, it was deleted for the LamB gene, and Top10 to amplify banks and methylate our mutated DNA. Indeed, MC1000 is endA+.
MC1000: F-, Δ(araA-leu)7697, [araD139],B/rΔ(codB-lacI)3,galK16,galE15(GalS), λ-,e14-,relA1, rpsL150(strR), spoT1, mcrB1
TOP10: F–, mcrA Δ(mrr-hsdRMS-mcrBC), Φ80lacZΔM15, ΔlacX74, recA1, araD139 Δ(ara leu) 7697, galU, galK ,rpsL (StrR) ,endA1, nupG
LB growth media :
You need :
Tryptone
Yeast extract
Agar
NaCl
To prepare 400 mL of LB media :
Add 4 g of Tryptone
Add 2 g of yeast extract
Add 4 g of NaCl
Add 8 g of Bacteriological Agar
Adjust to 400 mL with H2O milliQ
LB selective media :
You need :
Standard LB media
Vancomycine (Vanco)
Ampicilline (AMP)
Kanamycin (KAN)
Proline (Pro)
Ni or Zn if needed
For a standard petri dish :
In a falcon, add 20 mL of LB media
Add 66.67 µL of Vanco (3 mg/mL)
Add 20 µL of AMP (250 mg/mL)
Add 10 µL of KAN (50 mg/mL)
Add 25 µL of Ni or Zn (25 mM)
Add 0.743 µL of Pro (13.5 M)
M63 basic media :
You need :
(NH4)2SO4 , KH2PO4 , FeSO4.7H20 , MgSO4 , KOH
Noble Agar
Vitamin B1 (thiamin)
Carbon source
Amino Acids
For 400 mL of M63 Media :
Add 0.8 g of (NH4)2SO4
Add 5.44 g of KH2PO4
Add 0.2 mg of FeSO4.7H20
Add 8g of Noble Agar
Adjust the volume to 400 mL with H20Q (milliQ water)
Heat and mix until complete dissolution
Adjust to pH 7 with a solution of KOH
Sterilize in the autoclave
Complete the media with those elements :
400 µL of a solution MgSO4 1M (finale concentration : 1mM)
4 mL of a solution with the desire carbon source
- Solution 20 % industrial Maltohexaose (Conc. Finale : 0,2 %)
- Solution 20 % Maltotetraose Purified (Conc. Finale : 0,2 %)
- Other sources …
40 µL of a solution 0,5 % in Vitamin B1 (or a Thiamin solution)
Add 2 mL of a solution in desired amino acids :
- Solution Ile/Leu at 50/100 mM (Finale Conc. : 0,25/0,5 mM)
- Other aa …
NB : All the solutions have to be sterilized by filtration
Conserve at RT until use.
M63 selective media :
You Need :
M63 Basic media
Ampicilline (AMP)
Kanamycin (KAN)
Proline (Pro)
Ni or Zn if needed
For a standard petri dish :
In a falcon, add 20 mL of M63 Basic media
Add 20 µL of AMP (250 mg/mL)
Add 10 µL of KAN (50 mg/mL)
Add 25 µL of Ni or Zn (25 mM)
Add 0.743 µL of Pro (13.5 M)
Preparation of electrocompetents cells :
You need :
An O/N precultures
LB basic media
Antibiotics (if needed)
Sterile erlenmeyer
Some eppendorfs
Inoculate 400 mL of LB with 5mL of the O/N culture
incubate at 37°C (180 rpm) until reach a DO (600 nm) of 0.5-0.7
Centrifuge (4000 rpm, 4°C, 10 min)
Resuspend each pellet in 500 mL of sterile milliQ H2O (4°C)
Centrifuge (4000 rpm, 4°C, 10 min)
Resuspend each pellet in 250 mL of sterile milliQ H2O (4°C)
Centrifuge (4000 rpm, 4°C, 10 min)
Resuspend each pellet in 20 mL of sterile Glycerol 10% (4°C)
Transfer in eppendorfs
Centrifuge (4000 rpm, 4°C, 15 min)
Resuspend each pellet in 500 µL of sterile Glycerol 10% (4°C)
Aliquot cells ( 50 µL) and freeze them in liquid Nitrogen
Stock electrocompetent cells at -80°C
Electroporation :
You need :
DNA bank sample
Electrocompetent cells
Electroporation cuvette
Liquid LB, twice concentrated (LB 2x)
Sucrose solution 40% w/v
Each step should be done in cold environnement (ice bath) to slow down cells metabolism until incubation.
Inject 2µL of DNA sample in a 1.5mL Eppendorf tube filled with ~50µL of electrocompetent cells solution.
Rest for 20 minutes in the ice.
Prepare 1mL of “rescue medium” : mix 500µL of sucrose solution with 500µL of LB 2x.
Inject electrocompetent cells into an electroporation cuvette.
Place it into the electroporation device and give it a discharge of XkV.
Quickly inject the rescue medium in the cuvette and mix it with the cells.
Transfer the medium from the cuvette to a clean Eppendorf tube.
Incubate the tube at 37°C for ~2h.
Miniprep
We used the Sigma-Aldrich GenElute Plasmid Miniprep Kit.
Polymerase Chain Reaction (PCR) :
For the PCR reactions, we decided to work with the high fidelity Phusion Hot Start II DNA polymerase capable of amplifying long amplicons of genomic DNA.
PCR master mix (50µl total):
- 2,5 µl/primer
- 0,25 µl phusion
- 10 µl buffer
- 3 µl diluted template
- 1 µl DNTPs
- 30,75 µl H2O
