Team:Toulouse/Notebook/Protocols

1) Colony PCR
- Add 0.5µL of plasmid + 25µL of DreamTAQ MasterMix + 2µL of each 10µM primer (VR and VF2) + H20 qsp 25µL and take a colony.
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.
The following cycles have been used :
- 94°C - 5 min
- (94°C 45sec ; 55°C 45 sec ; 72°C 1min/kb ) * 25 cycles
- 72°C 5min
- Then 4°C

2) 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 2µL of plasmid + 2µL of Fast Digest Green Buffer + 1µL of each enzyme + Milli-Q water qsp 20µL
- Wait 15 minutes at 37°C and put the mix on a 1% or 2% gel for 30 minutes at 100V.

3) Sequencing The sequencing of the genetic constructions was performed by Eurofins Genomics Company by mixing 15µL of pure plasmid solution with 2µL of one primer.

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.