Experiments

Protocols

PCR

For the linearisation, we wanted to do a PCR:

PCR mix :

- 1µL primer Plasmid pSEVA438 diluted in 9µL of H2O (10 ng/µl)

- 25 µl Master mix 1X containing the Taq polymerase and nucleotide

- 2,5 µL of each primer
Gently mix the components by pipetting up and down or by briefly spinning the tubes/plates in a centrifuge. Program the thermal cycler with the appropriate temperature settings:

-initial denaturation: 98°C for 3 minutes (1 cycle)

-Denaturation: 98°C for 30 seconds (30 cycles)

-Annealing: 50°C for 30 seconds (usually 25-35 cycles; temperature depends on primer Tm)

-Extension: 72°C for 5 min (30 cycles; time depends on the length of the DNA fragment);

-Final extension: 72°C for 10 minutes (1 cycle)

Hold: 4°C

Integration of the Tn7 transposon by conjugation

Day 1 Inoculation of the four strains
On an antibiotic-free LB plate, spread successively (one on top of the other as far as possible, preferably with a swab) the four strains: P. putida KT2440 (recipient strain), E. coli PIR2 pBGXX (donor strain), E. coli DH5α-λpir pTnS-1 (transposase driver strain), E. coli pRK600 (helper strain). Incubate at 30°C overnight.
Day 2 Transplanting and selection
Transplant several strains grown overnight on cetrimide agar + 30mg/L gentamicin. Incubate at 30°C overnight.
Day 3
Resuspend a colony from the previous day using a toothpick, spread on LB plate (one colony / plate) + 30mg/L gentamicin. This can be done for several different clones in parallel. Incubate at 30°C overnight.

Pseudomonas culture

Pseudomonas putida KT2440 were grown either in LB broth suspension (10g/L peptone, 5g/L yeast extract, 5g/L sodium chloride) in Erlenmeyer flasks with incubation at 30°C at 150 rpm, or on cetrimide agar (20g pancreatic gelatin hydrolysate, 10g dipotassium sulfate, 1.4 magnesium chloride, 0.3g cetrimide, 13.1g agar in 1L water) at 30°C. Cultures of rhamnolipid-producing strains were grown with 10g/L D-glucose.

SDS-PAGE

Gel separation SDS-PAGE 12 %

- 1,6 ml Acrylamide-Bisacrylamide 29:1

- 1,3 ml H2O

- 0.04mL SDS 0.1%

- 1 ml Tp Tris pH 8.8

- 40 µl APS

- 1,6 µl TEMED
After the gel has polymerized, the gel concentration solution is prepared as follows:

- à,34 ml Acrylamide-Bisacrylamide 29:1

- 1,36 ml H2O

- 0,04 mL SDS 10%

- 0,25 mL Tp Tris pH 6.8

- 20 µl APS

- 2,5 µl TEMED
Before loading the samples: Add SB 4X -> Heat for 5 min at 90°C -> Centrifuge -> Load
The samples then migrate in the gel for 1 hour at 110 V. Once the migration is complete, the gel is stained with Coomassie Blue.

Plasmid construction using the Gibson assembly method

I. Enzymatic digestion
Enzymatic digestion involves two restriction enzymes, EcoRI and XmaJI, generating cohesive 5' outgoing ends. EcoRI cuts specifically at GAATTC sites and XmaJI at CCTAGG sites. With Fast Digest buffers, cutting times do not exceed 30 minutes at a temperature of 37°C. If required, Fast Digest buffer kits also contain FastDigest Green for staining digestion products on gels. Unfortunately, as we don't have an inactivation kit or elution means at our disposal, and the XmaJI enzyme is not heat-inactivatable, we won't be able to inactivate the restriction enzymes, at least not completely. This is likely to affect the quality and yield of the assembly.

Preparing the digestion solution with a total of 20µL, add the reagents in order at room temperature:

- 14µL nuclease-free water
- 2µL FastDigest 10X buffer (or Green Buffer if required)
- 2µL DNA (with a mass between 1-2.5 µg)
- 1µL of each of the two enzymes.
- Shake gently and incubate at 37°C for 30 minutes.

If in doubt, consider heat inactivation (at least for EcoRI). To do this, incubate the sample for 10 minutes at 65°C.
II. Fragment insertion
Once the plasmid has been digested, the ends left cohesive at 5' by enzymatic digestion are located downstream/upstream of the 40bp adaptor. A Gibson assembly can be performed directly after enzymatic digestion, and the t5 exonuclease contained in the master mix is supposed to digest the cohesive 5' ends to release the adaptor that will enable assembly of the plasmid with the fragments. There is one fragment per gene, i.e. 4 fragments and one plasmid, for a total of 10,861 bp. The protocol remains the same as in the instructions for use:

- In 20µL of master mix, add 50ng/µL of plasmid and 150ng/µL(x3) of fragment, equivalent to 1µg of plasmid and 3µg of fragment for a 1:3 ratio.
- Incubate at 50°C for 3h30*.
- Store at -20°C if necessary.
*Incubation time is based on the NEBuilder manual, which recommends 60 minutes for a 3180bp construct, i.e. 53bp/minute.
III. Bacterial transformation
Before transformation, the bacteria must be made competent. The first step is to obtain culture samples with a D.O. approximately equal to 0.8 in 25mL of culture in a growth phase.

To do this :
- Add 7mL of a KT2440 culture to 50mL of LB and incubate until O.D 0.8 (I had an O.D of 0.6).
- Centrifuge bacteria at 5000g for 10 minutes.
- Wash twice (recentrifuge in between), resuspending the pellet in 10% ice-cold glycerol.
- Resuspend cells in 400µL of 10% ice-cold glycerol.

Now competent bacteria can be transformed by electroporation:
- Add 1µL plasmid DNA to 40µL electrocompetent cells. Homogenize by gently mixing with a pipette several times. Transfer the mixture to a refrigerated cuvette.
- Wipe any moisture from the cuvette and insert into the device.
- Set the electroporator to Prokaryote "O" mode with a voltage of 2400V and a time constant of 5ms.
- Immediately add 1mL of LB and incubate for 2h at 30°C.
- Grow transformed bacteria on cetrimide plates with 30mg/L streptomycin (or other antibiotics).

Agarose gel electophoresis

Prepare a 1% agarose gel.
Pour the gel and add few drops of DNA stain:
Allow the gel to solidify for about 20-30 minutes
Mix the plasmid samples with an appropriate volume of loading buffer
Place the gel tray in the electrophoresis chamber filled with a TBE buffer.
Load the plasmid samples and a DNA ladder into the wells using a pipette.
Run the electrophoresis:
Apply a constant voltage (typically 100 V) across the gel using the power supply.
Run the gel for about 30 minutes.
Visualize the DNA

Rhamnolipids production test : Sheep blood agar

Mono-rhamnolipid-producing clones can be identified on cetrimide blood agar plates (7.5% (v/v) sheep's blood). Suspend a colony from day 3 plate. Inoculate a plate with cetrimide-blood agar + 30mg/L gentamicin, placing 1-2µL of the suspension so that drops are spaced far apart. Incubate overnight at 30°C. If mono-rhamnolipids are released into the medium, a halo should form around the competent colonies. Replicate colonies with the largest halo and keep them in the incubator at 30°C as stock.

Rhamnolipid production test - Methylene blue staining

This is a fast, simple method for analyzing rhamnolipids by complexing them with methylene blue. The method is based on measuring the absorbance (at 638 nm) of the rhamnolipid-methylene blue complex distributed in the chloroform phase.
Caution: this protocol uses chloridric acid and chloroform, so these products must be handled with gloves, goggles and a smock, in a chemical hood and using only suitable glassware.
Step 1 : From cultures lasting at least 24 h in the presence of glucose, recover the supernatant by centrifugation. From the supernatant obtained, fix the pH of the sample at 2.3 with HCl (1N). Transfer to a glass sample tube with a stopper.
Step 2 : Add chloroform on top of the supernatant to a volume 5 times the sample volume, the supernatant and chloroform phase will separate.
Step 3 : Remove 4mL of the chloroform phase and add to a methylene blue solution, the composition of which is detailed below*.
Step 4 : Mix vigorously for 4 min and leave to stand for 15 min.
Step 5 : Extract the chloroform phase and add to a glass/quartz cuvette to measure absorbance at 638nm. Do not forget to make the blank with chloroform.
*Methylene blue solution: Add 200 μL of 1 g/L methylene blue reagent and 4.9 mL of distilled water. The pH of this aqueous methylene blue solution should be pre-adjusted to 8.6 ± 0.2 by adding a 50 mM borax buffer.
Here is thelink to the article from which we derived our protocol

Protocol for inducing expression of the four genes using the XylS/Pm system

After identifying a clone that carries the plasmid with all four genes under the control of the Pm :
I. Induction and over-induction
Three different strains will be tested → 1. A WT KT2440 strain 2. A strain modified to produce rhamnolipids 3. A strain modified to produce rhamnolipids and sophorolipids.

Three cultures will be made to test three conditions for each strain (9 cultures in all) → 1. Without over-induction 2. With 3methyl-benzoate 1mM 3. With 3'methyl-benzoate 2mM.

For each condition: take a clone and culture in suspension in 10mL liquid LB + 30 mg/L gentamicin for rhamnolipid strains + 30 mg/L streptomycin for sophorolipid strains. Incubate at 30°C until D.O 600nm 0.100 is reached. For over-induction conditions, add 3methyl-benzoate to reach 1mM or 2mM depending on conditions. Incubate all cultures at 30°C for 4h.

Conditions	WT KT2440	WT KT2440	WT KT2440	RL	Rl	RL	RL + SL	RL + SL	RL + SL
Antibiotic	NO	NO	No	genta	genta	genta	genta + strepta	genta + strepta	genta + strepta
Toluene	No	1mM	2mM	NO	1mM	2mM	NO	1mM	2mM

Here is thelink to the article from which we derived our protocol

II. Protein extraction Prepare 50mL lysis buffer:

	Cf	Volume
NaP (pH 7.5) 0.2M	20mM	5 mL
NaCl 3M	500mM	8,3mL
QSP H2O MQ		50mL

- After removing the supernatant from the cultures, recover the pellet with the lysis buffer.
- Add 500µL lysozyme and incubate 15min on ice.
- Sonicate 11 times with 10 one-second pulses (parameter 40% amplitude and active cycle 50%).