Team:Aix-Marseille/protocols

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Protocols


1) Medium and solution protocol
Way to use it Compound name Protocol
DNA migration Agarose gel 1% 1g/100ml of agarose into TAE 1X
Supercompetant Escherichia Coli sp. cells Tfb1 30mM KOAc, 0.05M MnCl2, 10mM CaCl2, Glycerol 15%
Supercompetant Escherichia Coli sp. cells Tfb2 0,01M MOPS (pH=7), 0.075M CaCl2, 0.01 KCl, Glycerol 15%
SDS PAGE Loading buffer 2X 200 mM Tris HCl (pH = 6.8), 4% SDS, 0.02% Bromophenol blue, 20% glycerol, 2.5% βME
Electrophoresis buffer (10X) ( pH= 8,3) 0.025 M Tris, 0.19 M glycine, 0.1% SDS
Discolouring (200ml) 100 ml ethanol 96%, 20 ml CH3COOH, 169 ml water
Coomassie Blue (1L) 1.2 g Coomassie blue, 250 ml ethanol 95%, 80 ml acetique acide
SES 4X 50% glycerol, 20 mM EDTA, 1% SDS, 0.05% Bromophenol blue, 0.05% xylene cyanol
Western Blot Rouge Ponceau (100ml) stored in dark 0.2 g Ponceau S, 3% tricloroacetique acide
2) Molecular biology

A) Supercompetant Escherichia Coli cells (+/- 80 tubes of competent cells)

  • Streak E.coli cells on an LB agar plate with or without antibiotics.
    • 37°C, overnight.
  • Inoculate 3-4 colonies in 5 mL LB media (+ antibiotics selection if necessary),
    • 37°C, overnight.
  • Dilute the preculture to 1/100 in 200mL LB final(+/- OD600 = 0,05)
    • 37°C, 250 rpm, until OD600 = 0,5 (+/-2 hours).
    • Place cells on ice for 20 minutes.
  • Centrifuge cells in Sorval GSA rotor : 4°C, 10 minutes, 3500 rpm.
  • Discard supernatant
  • Cells must remain cold for the rest of the procedure (every steps in ice)
  • Discard the supernatant and resuspend cells in cold Tfb1 (30mM KOAc, 0,05M MnCl2, 10mM CaCl2, Glycerol 15%).
  • Centrifuge cells using Sorval RT6000B rotor : 4°C, 5 minutes, 3500 rpm.
  • Discard the supernatant et resuspend cells (by pipetting) in cold Tfb2 (1/10 volume of Tfb1) (0,01M MOPS pH=7, 0,075M CaCl2, 0,01 KCl, Glycerol 15%).
  • Incubate on ice for 15 minutes.
  • Transfer 120μL of cells into (1,5mL) Ependorff tubes placed on ice.
  • Cells stored at -80°C can be used for transformation for up to about 6 months.>

Additional steps :

  • Check the competence of bacteria by transformation (following “E. coli sp. Transformation protocol)

B) E. coli sp. transformation protocol :

  • 1 ng of DNA plasmid into a tube of super competent cells
  • 20 minutes in ice
  • Heat shock : 1 minute at 42°C
  • 5 minutes in ice
  • Adding LB medium to Vf = 1 mL
  • Shake : 1h, 37°C
  • Centrifuge : 4000 rpm, 4 minutes
  • Streak on agar plate (different dilution can be made)

C) Digestion/ligation cloning

Plasmid and insert digestion :

    • DNA (plasmid or insert) : 1μg
    • Enzyme 1: 2%
    • Enzyme 2: 2%
    • 1X buffer
  • 37°C, 3H (depend of restriction enzyme used)
  • PCR Clean up

Ligation :

    • T4 ligase 5%
    • Buffer T4 1X
    • Vector : 30 ng
    • Insert : 100ng
  • 1h, room temperature

D) SLIC cloning

Protocol optimised by Gauthier (GDP) (13/10/2020)

Excel

  1. Vector digestion during 1H : 2 µg
  2. Insert amplification : by PCR Q5
  3. Mix pour 50 µL :
    • Oligo1 : 2,5 µL
    • Oligo2 : 2,5 µL
    • dNTPs : 1 µL
    • 5xBuffer : 10 µL
    • GC Enhancer : 10 µL
    • DNA matrice : 1 µL
    • Eau : 22,5 µL
    • Q5 polymérase : 0,5 µL
    Protocol :
    • 98°C : 30 sec
    • 98°C : 10 sec
    • Tm°C : 30 sec
    • 72°C : 15 sec (pour 500 bp) go to #2 30 cycles
    • 72°C : 30 sec
  4. PCR Clean Up of insert(s) and restricted vector(s)
  5. Assembly of the cloning (s) and the control IN PCR TUBES (250 µL or 500 µL) with :
  6. a : See SLIC calculator file
    Cloning Control
    Vector restricted 0,03 pmola 0,03 pmola
    Insert 0,09 pmola 0a
    NeB Buffer 2.1 1 µL 1 µL
    T4 DNA polymérase 0,2 µL 0,2 µL
    H2O ppi Qsp 10 µL Qsp 10 µL
  7. Incubate between 2 minutes 30 secondes and 3 minutes at room temperature
  8. Incubate 10 minutes in ice
  9. Adding 100 µL of competent cells and perform a classic transformation protocol.
    1. 1 h in ice
    2. 2 minutes at 42 °C
    3. 5 minutes in ice
    4. 5 minutes at room temperature, adding 900 µl 2YT
    5. 1 h at 37 °C
    6. Spread on medium + Ab, 100 µl and all the rest (centrifuge 5 min at 5000 rpm, remove 850 µl and resuspend with the remaining 50).

E) Megapriming

Mix PCR:

  • 2,5 µl 10X reaction buffer
  • 0,5 µl dsDNA template
  • 1 µl primer 1 (10 µM)
  • 1 µl primer 2 (10 µM)
  • 5 µl dNTP (2.5mM)
  • qsp H2O 24 µl
  • 1 µl Pfu Turbo DNA polymerase (ou Pfu Ultra II)

Program:

  • 30min, 95°C
  • 16 cycles
    • 95°C, 30min
    • 55°C, 1 minute
    • 68°C, 1 to 2 minutes/kb of plasmid
  • Then, following the transformation protocol
3) Biochemistry

A) Overproduction : optimized protocol (pBAD24 induction)

  • Bacteria preculture in LB medium with antibiotics : All day, 37°C
  • Back diluted 1/100 in new LB medium with antibiotics
  • 37°C until OD600nm = 0.4
  • Induced with [Arabinose] = 1%
  • 28°C, overnight

B) Westernblot protocol

  • Following the overproduction proteins
  • Take 1UDO and adding 20µL of loading buffer 2X
  • 10min, 95°C
  • 2x SDS PAGE gel migration : +/- 45min, 200V in YZ buffer 1X
    The second SDS PAGE gel can be revealed into Bleu coomassie coloration
  • Western Blot : Transfert (Blot), construction
    • 2x Wathman (previously soaked in transfer buffer)
    • Nitrocellulose (pre-soaked in transfer buffer)
    • 1x Gel SDS PAGE
    • 2 xWathman (previously soaked in transfer buffer)
    • 30 to 45 min, 80V (depend of the protein size)
  • Western Blot : Antibody revelation (following the company protocol)
    • Blocking
    • First antibody
    • Washes
    • Second antibody
    • Revelation (depend of secondary antibody)

C) Purification protocol : strep tactin II column

  • Strep-Cyt1Aa overproduction : Following the “Overproduction : optimized protocol (pBAD24 induction)” protocol. The protocol was done with 2 litters of LB medium.
  • On morning : DO 600nm measurement
  • Centrifuge cells : 5000g, 20min, 4°C.
  • Resuspend the pellet with 10mL of PBS 1X
  • Centrifuge cells : 5000g, 20min, 4°C.
  • Discard the supernatant

Cells lysis :

  • Resuspension of the pellets in 10 mL of buffer W (buffer for Strep-Tactin purification column)
  • Adding lysozyme (1mg/mL)
  • 37°C, 30min
  • Adding Protease inhibitor 1X and Dnase 1 (0.1mg/ml)
  • Adding MgCl2 (10mM)
  • 20min, room temperature
  • Destroy cells using french-press
  • Ultracentrifugation : 45 000g, 1H

Proteins purification (using Strep-Tactin Purification column)

  • For 1 liter of culture: put 1 ml of the bead mixture on the column
  • Balance the balls: 3 passes of 5 volumes of column (CV) of buffer W
  • Adding lysat (supernatant after ultracentrifugation) with a ball
  • Incubate 1H, 4°C, on the balance
  • Pass all of the lysate through the column
  • Washes 5 fois, 5CV tampon W
  • Elution :
    • Adding 500µL of elution buffer
    • Incubate 20min à 4°C
    • Adding 1ml of elution buffer (repeat 2 times)
  • Washing the column :
    • 5-10mL of H2O
    • 5mL NaOH 1mM
    • 5ml H2O
    • 5ml Ethanol 20%
    • Keep the column in ethanol , conserved at 4°C
  • Doing SDS PAGES and Western Blot analysis

D) Timer lysis device experimentation

  1. Test timers: objective 1 (To determine if mitomycinC has an effect -dose or effect on/off. Choice of the culture DO and dose of mitomycinC for subsequent tests).
    • Preculture strains MG1655 pRL -GFPcolA, MG1655 pRL -lysRFP and MG pRL1 LB medium with antibiotics
    • 37°C, overnight
    • Back Dilution at OD 0.01, culture 25 mL at 37°C with agitation. (Two DOi to test: phase expo DO=0.4 and phase stat DO=1.)
    • We OD600nm is 1 or 0.4 : culture into 5 erlens of 5 mL (i.e. 15 erlens).
    • Induced gene : Addition of [mitomycin C]: 0, 50, 300, 500 and 800 ng/mL.
    • Incubation at 37°C.

    Every 30 min for 5 hrs take 200 uL sample for measuring : DO600, fluoGFP and flouGFP (reading with a plate 96 black wells at TECAN)

  2. Test timers: objective 2 (follow the” accumulation of PFM and PFR in the course of time).
    • Preculture strains MG1655 pRL1, MG1655 pRL -GFPcolA and MG1655 pRL -lysRFP in LB medium with antibiotics
    • 37°C, overnight
    • Back Dilution to OD 0.01, culture 50 mL at 37°C with agitation. (Two DOi to test: phase expo DO=0.4 and phase stat DO=1.)
    • At DOi expected, separate cultures into 2 erlens of 20 mL: an untreated control and a mitomycin C treated: [mitomycin C] = 800 ng/mL.
    • Incubation at 37°C for 5 hours.

    Every 30 min.

    1. Take : 200 µL sample : reading fluoGFP and flouGFP (reading with a plate 96 black wells at TECAN)
    2. Sample of 1 mL : centrified for 5 min at 8000 rpm. Sampling of SN for samples induced.
      • Cap + 40 µL charging buffer. vortex. 5 min at 95°C and then ice.
      • 1 mL SN + 10% TCA (trichloroacetic acid).
      • 30 min on ice, centrifugation for 20 min, 14 000 rpm and remove SN
      • add 200 µL acetone 100%.
      • Centrifugation: 15 min 14 000rpm
      • Take all the SN. Allow the pellet to dry in the oven at 37°C
      • Resume in 40µL of charge buffer.
    • SDS PAGE gel migration analysis : Cap uninduced / Cap induced/induced SN (for each sample)