Team:Toulouse/Notebook/Protocols

E. coli competent cells

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
- 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

E. coli transformation protocol

- 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.

Miniprep and alcaline lysis

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%

Cloning

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

First Step
BOTH PARTS HAVE THE SAME ANTIBIOTIC RESISTANCE
1) Digestion mix
For the vector :
- 5 µL of miniprep plasmid
- 2 µL of each restriction enzymes
- 2 µL of Green Buffer
- 9 µL of Milli-Q water

For the insert :
- 10 µL of miniprep plasmid
- 2 µL of each restriction enzymes
- 2 µL of Green Buffer
- 4 µL of Milli-Q water
- Incubate 15 minutes at 37°C

2) Gel extraction
- Prepare a 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 for 30 min at 100V or 1hour at 50V.
- The revelation is made in BET (10minutes) and then 5minutes in water.
- The gel extraction is realized thanks to the THERMO SCIENTIFIC GeneJET Gel Extraction and DNA Clean Up Microkit.

3) Inactivation of the enzymes for the vector
There are two ways to inactivate the enzymes :
- Use of DNA Clean up kit for the DNA fragment above 200 pb
- Heat inactivation at 95°C for 10 minutes.

THE TWO PARTS HAVE A DIFFERENT ANTIBIOTIC RESISTANCE
1) Digestion mix
For each part, add :
- 5 µL of miniprep plasmid
- 1 µL of each restriction enzymes
- 2 µL of Green Buffer
- 9 µL of Milli-Q water
- Incubate 15 minutes at 37°C

2) Inactivation of the enzymes for the vector
There are two ways to inactivate the enzymes :
- Use of DNA Clean up kit for the DNA fragment above 200 pb
- Heat inactivation at 95°C for 10 minutes.

Second step
Ligation
- Mix 10 µL of insert + 4µL of vector + 2µL of 10x T4 buffer + 0.5µL of T4 ligase + 3.5µL of Milli-Q water
A control without insert must be made
- Incubate the ligation mix 15 minutes at room temperature (22°C) and keep the tubes in ice or at -20°C to prepare the transformation

2) Transformation
- Take 5µL of the ligation mix for 50µL of competent cells and use the Toulouse iGEM Team 2014 transformation protocol.
- Plate the solution on selective medium overnight at 37°C.

Checking of the genetic constructions

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.

B. subtilis transformation

Strain: Bacillus subtilis 168.
Plasmid : pSBBS4S given by the Munich University iGEM Team. l’équipe iGEM de l’université de Munich. This plasmid is replicative in E. coli and integrative in Bacillus subtilis.

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 of B. Subtilis strain 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 for the aeration) and put 5µL of Miniprep DNA.
- 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.

Checking of the genetic constructions after plasmid integration in Bacillus subtilis

Test of the pSBBS4S plasmid integration in Bacillus subtilis genome on the threonine site:
- Plate the transformed Bacillus strain on a selective medium (LB + spectinomycin) overnight
- The obtained clones are then plated on different media: Medium Competence (Thr+), Medium Competence (Thr-) and LB + spectinomycine.
When the plasmid is integrated, the clone can grow on minimum medium without threonine but can not grow on the other media. Moreover, a colony PCR can be performed with the same protocol as previously presented. The used primers are VFBS and VRBS.

Final Tests

Chemotaxis test

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.

- Put 200µl of the different chemoattractants in the wells of the ELISA plate and pipette 15µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control 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 1/10,000 and 100µL is spread on LA medium.
- The plates are then incubated overnight at 37°C.

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

CAUTION : all the lab equipment must be desinfected before the manipulations with the fungi.
Three different funguss strains were used : Aspergillus brasiliensis, Aspergillus nidulans, Trichoderma reesei
- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.
- Then the drop is mixed with 1mL of sterile water.
- a microscopy count can be performed thanks to Toma cell to determine the conidia concentration.
NB : The conidia solutions are then diluted and spread on sap medium to get 10,000 conidia/plate.
- After 72hours of liquid culture of different clones of B. subtilis with the fungicides module, the culture can be centrifuged.
- 130µL of supernatant is used to soak a plug on a fungus plate. The bacterial pellet is resuspended in 130µL of LB medium and put on a tampon.
- The plates containing 10,000 conidia and the supernatant or bacteria soaked plugs are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wilt type strains or copper sulfate at 10 and 20mg/mL.

Fungicide test: in planta assay

The first step involves doing the inoculation of SubtiTree in plants through stomata (opened in wet condition). We dilute our bacterial samples for two concentrations: 5.10^6 and 10^8 bacteria per mL. The bacterial solutions of WT, having the expression cassette of the D4E1 or D4E1-operon GAFP1 are introduced into plants (a control test without bacteria was performed). Thanks to a 1 ml syringe (without needle),we inject plant with bacteria by pressure. We use five leaves of each plant, marked with a marker point. It is necessary to repeat the operation on both sides of the leaf and the excess is wipe off. The plants are placed in a growth chamber (phytotron) to control light, high humidity, temperature and bacterial non-proliferation. Compared for Agrobacterium species, bacterial growth in the plant is left for 24 h.
The next step begins with preparation of the fungal samples. PDA cores containing the fungal culture is crushed and left in culture in the PDB . Then it pass through a filter of 100 µm (to remove large aggregates) and through a 40 µm filter to allow small molecules. The caught hyphae are placed in the PDB for 24 to 48 hours. This liquid culture is filtered to retain the living fragment and place in solution. Then, 2.5 of OD is adjusted. Obtained leaves above are detached from the plants using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf is deposited 5μL of fungal solution (using beveled tips because it is too viscous), as control we keep inoculated leaf without fungus. Pictures are achieved at different times. All the plants are destroyed by autoclaving.