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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

Vector digestion during 1H : 2 µg
Insert amplification : by PCR Q5

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

PCR Clean Up of insert(s) and restricted vector(s)
Assembly of the cloning (s) and the control IN PCR TUBES (250 µL or 500 µL) with :

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

Incubate between 2 minutes 30 secondes and 3 minutes at room temperature
Incubate 10 minutes in ice
Adding 100 µL of competent cells and perform a classic transformation protocol.

1 h in ice
2 minutes at 42 °C
5 minutes in ice
5 minutes at room temperature, adding 900 µl 2YT
1 h at 37 °C
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

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)

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.

Take : 200 µL sample : reading fluoGFP and flouGFP (reading with a plate 96 black wells at TECAN)
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)

Team:Aix-Marseille/protocols

Protocols

1) Medium and solution protocol

2) Molecular biology

3) Biochemistry