Team:Toulouse/Notebook/Protocols

Day 0

- Make an Escherichia coli cell culture in LB medium overnight

Day 1

- Freeze 0.1 M CaCl2 and 4 Falcon tubes of 50mL at 4°C
- Streak 2% culture in LB to get an OD of 0.3 to 0.4 (it takes approximately 1h30)
- Centrifuge 10 minutes at 4500 RPM
- Remove the supernatant
- Resuspend the pellet in 7.5 mL of 0.1 M frozen CaCl2
 Freeze the glycerol
 Prepare 20 Eppendorfs tubes and put them in ice
- Centrifuge 10 minutes at 4 500 RPM
- Resuspend the pellet in 500µL of 0.1 M CaCl2
- Add glycerol for a final concentration of 15%
- Keep the tubes at -80°C

Time	Lab equipment	Preparation of media
3 hours 30 minutes	LB agar medium plate containing the proper antibiotic (2 plates per transformation), 1 tube of E. coli competent cell per transformation, 1,25mL of LB medium per transformation, water baths at 37°C and 42°C, sterile water for kit plate	LB medium : 10g/L Tryptone + 5g/L Yeast extract + 10g/L NaCl LB agar medium : 10g/L Tryptone + 5g/L Yeast extract + 10g/L NaCl + 15g/L agar

- Let the LB agar medium plates dry in a sterile area
- Thaw out the competent cell aliquotes for about 10 to 20 minutes
- Add 20 to 100 ng of plasmid or 3µL of kit plate DNA

NB: for kit plate, resuspend the well in 10µL of sterile water - Put the tubes 20minutes in the ice
- Put the tubes 2 minutes at 42°C in the water bath
- Put the tubes back in ice immediately to create the thermic shock
- Add 1mL of LB medium
- Put the tube 2 hours in the 37°C water bath (1hour if it concerns an ampicillin resistant strain) to allow the phenotypic expression
- Centrifuge for 1 minute at 13 000 RPM
- Remove the supernatant
- Resuspend in 250 µL of LB medium
- Streak the final mix on LB agar selective medium: 200 µL on one plate, 50µL on the second plate.

Time	Lab equipment
1 night + 1 hour	Plates after transformation, culture tubes, LB (5mL x number of tubes), QIAprep spin Miniprep Kit n.27106., cryotubes, Eppendorf tubes

Day 0

- Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C…)
- Resuspend one colony/culture tube in 5mL of LB medium with antibiotic
- Leave the culture shakes overnight at 37°C

Day 1

- Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with a hot elution buffer or water at 55°C.
- Keep the tubes at -20°C

NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200µL of buffer 1 is added to resuspend the pellet, 400µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM. 60µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse.
Buffer 1: Tris 10 mM pH 8 + EDTA 1mM
Buffer 2: NaOH 2mM + SDS 1%
Bufer 3: A COOK 3M + A COOH 15% - Try the best strain on PCR colony and digestion
Digestion mix with a 20µL final volume:
 2µL of plasmid
 2 µL of green buffer
 1 µL Enzyme 1
 1µL Enzyme 2
 14 µL miliQ water
- Cryogenise the best colonies in cryotubes : 800 µL of the culture + 200µL of pure glycerol
- Cryotubes are marked with the bacterium name, the name and the number of the biobrick and the date.
- Each tube has a number on the top to identify easily in the freezer at -80°C

Time	Lab equipment
2 days	Competent cells, BioBricks transformed, Miniprep kit, restriction enzymes, agarose gel (1 or 2%), waterbath, T4 DNA ligase, Eppendorf tubes

After taking the competent cells, transforming the Biobricks and making the miniprep, make the digestion mix.

BOTH PARTS HAVE THE SAME ANTIBIOTIC RESISTANCE
1) Digestion mix
- 10 µL of miniprep plasmid
- 2 µL of each restriction enzymes
- 2 µL of Green Buffer
- 6 µL of water
 Incubate 15 minutes at 37°C

2) Make an electrophoresis
- Prepare 1% of 2% electrophoresis agarose gel with 0.5x TAE buffer
- Put 20µL of sample + 6µL of marker (1kb for 1% gel and 100pb for 2%) into the well
- Migration 30 min at 100V
- The revelation is made in BET (10minutes) and then 5minutes in water

3) Gel extraction with Thermofisher kit
- Remove quickly under UV the DNA fragment from BET to avoid any kind of mutation. Place the fragment in a 1.5mL Eppendorf tube. Make sure to excise as close as possible from the fragment. Take a picture under UV before and after the excision.
- Add 200µL of extraction buffer and mix everything
- Incubate 10-15 minutes at 50-58°C until the gel is dissolved completely.
- Add 200µL of ethanol and mix
- Transfer into a column tube and centrifuge 1minute at 14 000 g and remove the liquid
- Add 200µL of Prewash buffer and centrifuge 1minute at 14 000 g and remove the liquid
- Add 700µL of Wash Buffer and centrifuge 1minute at 14 000 g and remove the liquid. Repeat this step twice.
- Centrifuge the tubes without any liquid 1minute at 14 000 g - Transfer the column in another 1.5mL Eppendorf tube
- Add 10µL of Elution Buffer and centrifuge 1minute at 14000 g - Remove the column and keep the tubes at -20°C

THE TWO PARTS HAVE A DIFFERENT ANTIBIOTIC RESISTANCE
1') Digestion mix
- 2 µL of miniprep plasmid
- 1 µL of each restriction enzymes
- 2 µL of Green Buffer
- 14 µL of water
 Incubate 15 minutes at 37°C

2') To inactivate the enzymes, use a PCR clean up kit or a heat inactivation treatment at 95°C for 10min

3') Proceed to the ligation

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4) Ligation
- Mix 8µL of insert + 2µL of vector + 2µL of 10x T4 buffer +0.5µL of T4 ligase +H20 qsp 20µL
A control without insert must be made
- Incubate 10 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation

5) Transformation
- Take 5µL of the ligation mix for 50µL of competent cells and use the transformation protocol.
- Plate the mix on selective medium overnight

6) Colony PCR
- Take a colony and add 0.5µL of plasmid + 25µL of DreamTAQ MasterMix + 5µL of 10µM primer + H20 qsp 50µL.
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.

7) Analytic digestion
- Put a colony in 5mL of LB selective medium and wait for 6 hours
- Make a purification thanks to the Miniprep kit
- Mix 5µL of plasmid + 1µL of Fast Digest Green Buffer + 0.3µL of each enzyme + water qsp 10µL
- Wait 45minutes at 37°C and put the mix on a 1% or 2% gel

Day 0

- Streak out the Bacillus strain and plate this on an LB agar plate overnight at 37°C

Day 1

- Pick up a nice big colony and drop it in 2 ml of completed 1x MC
- Grow at 37°C for 5 hours
- Mix 400 µl of culture in a fresh tube ( tubes loosely closed – aeration) and put 6µL of DNA linearized with 1µL of ScaI restriction enzyme
- Grow the cells at 37°C for an additional 2 hours
- Spread the complete 400 µl reaction mix on selective antibiotic plates, and incubate at 37°C overnight

Preparation of solutions

300 mM Tri-Na Citrate:
- 0.88 g Tri-Na Citrate
- 10mL MQ water

Ferric NH4 citrate:
- 0.22g Ferric NH4
- 10mL MQ water

10x Competence Medium
For 10mL:
- 1.40g K2HPO4
- 0.52g KH2PO4
- 2g glucose
- 1 mL 300 mM Tri-Na citrate
- 0.1 mL Ferric NH4 citrate
- 0.1g Casein Hydrolysate
- 0.2 g Potassium glutamate
The complete mixture should be dissolved in 10 ml. First add 5 ml milliQ water and mix. When everything is dissolved add MQ water till 10 ml. Filter sterilize the complete mixture and store at -20°C.

1x Competence Medium
- 1.8 mL MQ water
- 200 µL 10x Competence Medium solution (previously filter sterilized)
- 6.7 µL 1M MgSO4 (previously autoclaved)
- 10 µL 1% tryptophan (previously filter sterilized and stored in aluminium foil)
The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.

Plasmid integration test in Bacillus subtilis
Test of the plasmid integration in Bacillus subtilis genome on the threonine site:
- Plate the transformed Bacillus strain on a selective medium overnight
- The obtained clones are then plated on different media: MC(Thr+), MC (Thr-) and LB. These media are equivalent to the ones used for the competence. But in MC(thr-), the casein hydrosylate is not inserted and replaced by NH4Cl (same amount), threonine is added (5mg/ml). (for the complete protocol, see transformation > B. subtilis) NB : These media are equivalent to the ones used for the competence except that :
- For MC(thr-), the casein hydrosylate is not inserted and replaced by NH4Cl (same amount), threonine is added (5mg/ml). (for the complete protocol, see transformation > B. subtilis)
-For 500ml of medium :
-- 450 ml Water
- 7,5g agar
-1,7 ml MgSO4 (1M)
-Autoclave
-Add 50ml of 10X MC (+/- thr) and 2.5ml of tryptophane (1%)

Binding test

CBB (Chitin Binding Buffer):
- 500 mM NaCl
- 20 mM Tris-HCl
- 1 mM EDTA
- 0,05% Triton X-100, 25°C, pH=8

Column activation:
- Vortex the beads
- Put 50 µL of beads in a 1.5mL centrifuge tube
- Wash with 500 µL of CBB
- Put the centrifuge tube on a magnetic rack
- Wait 30 seconds
- Remove supernatant
- Repeat the wash

Bacterial fixation on the chitin beads:
- Add 200 µL of bacteria solution (105 bactéria/mL)to the washed beads
- Shake during 1h at 4°C
- Add 500 µL of CBB (washing A)
- Put the centrifuge tube on a magnetic rack
- Wait 30 seconds
- Remove supernatant
- Add 500 µL of CBB (washing B)
- Put the centrifuge tube on a magnetic rack
- Wait 30 seconds
- Remove supernatant
- Add 500 µL of CBB to recover the beads directly

Bacteria count:
- Make different dilutions : 10-1, 10-3, 10-5 of the first bacterial culture and spread on LA plates
- Make different dilutions : 1, 10-2, 10-4 of washings (A and B) and of the beads in CBB medium and spread on LA plates
- Place the plates at 37°C overnight
- Count colonies on different plates

Fungicide test: anti-fungal activities

Time	Lab equipment	Preparation of media
2 days	Petri dishes with PDA ¼ medium, fungus, Eppendorf tube, sterile water, parafilm, Thoma cell, overnight culture of SubtiTree, tips to make the holes, LB + agar	PDA ¼ medium : 2.25 g Agar + 1.95 g PDA (39mg/mL) then complete with 200 mL of water  autoclave

1- Counting conidia
/!\ SAFETY : under the biosefty cabinet /!\
- In order to plate an exact number of conidia on PDA plate, first count the conidia  Place a drop of Tween 80 on the fungal plate, let stand (make it roll if possible) and take back the drop in a sterile Eppendorf tube filled with 1ml of water.
- Mix and vortex.
- Place a drop of the mixture in a Thoma cell and count the number of conidia.
- Make dilutions of the solution in order to get the right amount of conidia in each plate: 25.000 /plate and 5000 plate
NB: As we want to spread 200µl per plate you have to get a final concentration of 125.103 conidia / ml (and 25 000 conidia /ml, just make a dilution of the first solution to get this one).
- Spread on the PDA plate 200 µl of the conidial suspension.

2- Bacterial plugs
- Make 2 holes in each PDA plates with a sterile blue tip.
- Centrifuge 2ml of an overnight culture of SubtiTree.
- Resuspend the pellet in 1ml of LA (less to increase the concentration if you have less tests to perform)
- Pipet 200µl of the mixture in the hole previously made
 200µl / fungi / concentration of conidia (4 conditions are tested : Asp 25000, Asp 5000, Glo 25000 and glo 5000)
/!\ Add the LA at the very last minute, just before putting the mixture in the holes to avoid the solidification/!\
- Place the plates at 30° after putting parafilm around them
- Do not forget to test the WT and the copper sulphate!

3- Clean everything
- Decontaminate the biosafety cabinet (and everything inside) and let the disinfectant into the biosafety cabinet during 30 minutes!!

Chemotaxis test

Time	Lab equipment	Preparation of media
1 day	96 wells ELISA plate, Multichannel pipette	LB medium : 10g/L Tryptone + 5g/L Yeast extract + 10g/L NaCL

Many chemotaxis tests exist such as plate tests or capillary tests. We tried all of them and we decided to optimize the « Capillary essay » from Imperial College 2011 iGEM team.
- Prepare the bacteria in LB medium so they reach an OD between 0.5 and 0.6 (exponential growth phase). This step takes about 5 hours.
- Prepare the multichannel pipette: improve the cohesion between the tips and the pipette with Blu-Tack to avoid air and the possibility of leakage.

SCHEMA A COMPLETER

- Put 200µl of the different chemoattractants in the wells of the ELISA plate and pipette 15µL of each with the multichannel pipette: fushine which represents our negative control, tryptophan, glucose and N-acetylglucosamine (NAG). NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on Ceratocystis platani wall. The volume in the tips must be marked.
- Put the tips with chemoattractants in 300µL of the bacterial solution in exponential growth phase in the ELISA plate.
- Let the installation settle for 1 hour at room temperature.
- After an hour, put the volume of the tips on parafilm.
- Each solution is diluted 1/1000 and 100µL is spread on LA medium.
- The plates are then incubated overnight at 37°C.