iGEM 2023 — Team sorbonne-u-paris

### NOTEBOOK

### Engineering

The Engineering process is composed of four stages: Learn, Design, Build, and Test

## Learn

![](https://static.igem.wiki/teams/4643/wiki/learn.png)

Our Problem: Soil Contamination

Polycyclic Aromatic Hydrocarbons
(PAHs) are harmful pollutants commonly found in environments due to industrial activities,
urbanization, and natural processes. They pose significant risks to human health and the ecosystem,
as they are known to be carcinogenic and persistent in the environment.

Polycyclic aromatic hydrocarbons
(PAHs) present in soil have the potential to enter surface and groundwater systems through
processes like precipitation and surface runoff. These chemicals can also be released into the
atmosphere through volatilization. These pollutants subsequently find their way into crops through
root and leaf absorption from contaminated soil and air. This contamination could lead to the
accumulation of PAHs in various organisms, throughout the food chain until it reaches us, humans.

## Design

![](https://static.igem.wiki/teams/4643/wiki/design.png)

Our Solution:

By modifying *Pseudomonas putida*, a bacteria
that naturally thrives in soil environments, to enhance its metabolic pathways responsible for the degradation of PAHs into harmless byproducts,
we offer an innovative and eco-friendly approach to soil decontamination. This approach is both sustainable and environmentally friendly...

![](https://static.igem.wiki/teams/4643/wiki/beforepseudodetox.jpg)
![](https://static.igem.wiki/teams/4643/wiki/afterpseudodetox.jpg)

## Build

![](https://static.igem.wiki/teams/4643/wiki/build.png)

## Rhamnolipids Synthesis

During our research, we discovered that *Pseudomonas putida*
has been extensively studied for rhamnolipid production. We are grateful to Dr. Till Tiso for providing us with four different strains.

* *E. coli DH5α λpir pSK02* (containing the mini-Tn7-delivery plasmid with genes
  *rhlAB* under the control of the stacked synthetic promoter *ffg*).
* *E. coli HB101 pRK2013* (helper strain enabling the transfer of plasmid-DNA).
* *E. coli DH5α λpir pTNS-1* (helper strain providing the transposase).
* *P. putida KT2440*

We employed a bacterial conjugation protocol
to transfer the rhlAB genes to *P. putida* KT2440, enabling it to produce rhamnolipids.

## Sophorolipids Synthesis

We discovered that 4 enzymes were required for
the synthesis of sophorolipids :

* Cytochrome P450 CYP52 M1
* UDP-glucosyltransferase A1 (UGTA1)
* UDP-glucosyltransferase B1 (UGTB1)
* Lactonase (or Lactone esterase)

These four enzymes are naturally produced in eukaryotic cells,
such as in *Starmerella bombicola*. Our objective was to engineer *P. putida* to induce the synthesis of these four proteins, resulting in the production of sophorolipids.

To accomplish this, we reached out to Dr. Victor Delorenzo, a *Pseudomonas* expert.
After several meetings with him, we attempted to design a plasmid that would incorporate the genes for these diverse enzymes:

![](https://static.igem.wiki/teams/4643/wiki/plasmide-sophorolipid.png)

To create this plasmid, we requested Dr. De Lorenzo
to provide us with the pSEVA438 plasmid, which will serve as our backbone:

![](https://static.igem.wiki/teams/4643/wiki/pseva438.png)

Then we designed the 4 bricks, each containing a gene:

![](https://static.igem.wiki/teams/4643/wiki/cypbrick1.png)

![](https://static.igem.wiki/teams/4643/wiki/ugta1-brick1.png)

![](https://static.igem.wiki/teams/4643/wiki/ugtb-brick1.png)

![](https://static.igem.wiki/teams/4643/wiki/lactonasebrick1.png)

Using the Gibson assembly method, we combined these
different fragments to create the desired plasmid necessary to induce sophorolipids synthesis.
We transformed *Pseudomonas putida* with the newly constructed plasmid primarily through
electroporation, as well as using a heat-shock protocol.

## Test

![](https://static.igem.wiki/teams/4643/wiki/test.png)

## Testing Rhamnolipid Production / Insertion of Rhamnolipid Genes

1. Antibiotic Selection

![](https://static.igem.wiki/teams/4643/wiki/test-results-3.jpg)

*Pseudomonas putida* was initially the only strain expected to grow on cetrimide agar.
However, we discovered that *E. coli* DH5*α* wild type (WT) could also thrive in this medium.
The *P. putida* colonies exhibit distinct characteristics compared to the DH5*α* WT colonies,
making them easily distinguishable on the agar plate. The *P. putida* colonies are identifiable by
their green coloration. When gentamycin is introduced to the cetrimide plate, it effectively eliminates both
DH5*α* WT and *P. putida* WT, but the conjugating *P. putida* strain remains viable.
Upon further investigation, we encountered a tube of *E. coli* DH5*α* that could grow on cetrimide
with gentamycin. However, a colony comparison revealed that it was indeed *P. putida* that had contaminated
this *E. coli* DH5*α* WT tube. This indicates the success of our conjugation technique, as we have
managed to integrate mini-TN7 into the genome of *P. putida*.

The bacteria present on this plate are resistant to gentamycin, meaning they were successfully transformed (or at least partially since they acquired the resistance gene from the transforming plasmid).

2. Blood Agar Test:

![](https://static.igem.wiki/teams/4643/wiki/bloodagar.png)

No halo appears to have formed around any of the transformed *P. putida*> colonies—the presence of a halo would suggest the production of rhamnolipids.

3. Measurement of Rhamnolipids by Spectrophotometry

![](https://static.igem.wiki/teams/4643/wiki/dosage-controle.png)

![](https://static.igem.wiki/teams/4643/wiki/dosagerhamno.jpg)

The methylene blue assay method
was used to measure the concentration of rhamnolipids following the culture of
transformed *Pseudomonas putida* bacteria and extraction of its lipids. This involves measuring the absorbance of the rhamnolipid-methylene blue complex in a chloroform
phase (at 683 nm). Despite observing an absorption peak expected to correspond to rhamnolipids (on the right) resulting from the modified strain, the control strain yielded similar absorbance measurements.
This could be due to no or very limited rhamnolipid production, but it may suggest that this method might not be sensitive enough and may detect other extracellular lipids instead.

**4. Sequencing (Nano-pore)**: 50% alignment/identity with original transposon.

## Testing Insertion of Sophorolipids Genes

1. Antibiotic Selection:

![](https://static.igem.wiki/teams/4643/wiki/transfosophorolipids1.jpg)

We selected our transformed *Pseudomonas putida* for sophorolipids
with streptomycin. On the right : *P. putida* Wild Type (no bacterium). In the middle: *P. putida* transformed.
On the left: *P. putida* transformed.The transformation appears to have worked.

2. Protein expression:

![](https://static.igem.wiki/teams/4643/wiki/sophorolipidssds.png)

Protein expression was studied through SDS-PAGE electrophoresis of
transformed *P. putida* following inoculation with 0mM, 1mM, and 2mM salicylate over a 4-hour period.
Our gel was stained with Coomassie Blue, revealing distinct profiles between the electrophoresis results and
the transformed *P. putida*. The presence of bands within the 50 to 75 kDa range for
transformed *P. putida* suggests the existence of new proteins possibly associated with the transformation process.

### 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 the[link](https://link.springer.com/article/10.1007/s00253-009-1896-9)  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 the[link](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4225500/)  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%).

### Wet lab results

# Rhamnolipids synthesis :

* Conjugation :

After receiving our four strains, a donor E. coli strain with the plasmid carrying the Mini-Tn7 transposon, two helper E. coli strains, and the P. putida KT2440 recipient strains, we carried out conjugation for three days to obtain a KT2440 strain that had integrated the transposon carrying the gentamicin resistance gene and the two genes encoding the RhlA and RhlB proteins, enabling rhamnolipid synthesis by Pseudomonas putida. It should be noted that the transposon integrates specifically on the att sites of the bacterial genome, in an operon that carries genes involved in cell membrane biosynthesis and therefore constitutively expressed. Once conjugation is complete, gentamicin-resistant strains producing rhamnolipids are expected.

![](https://static.igem.wiki/teams/4643/wiki/schema-conjugaison.png)

![](https://static.igem.wiki/teams/4643/wiki/test-results-3.jpg)

**Figure 1:** Photo of our isolation medium with our different bacteria. On the left, these are the cetrimide plate and on the right these are the cetrimide + gentamicin plate. In the first row, these are the contaminated tube of E.coli DH5α (Pb) (left of the plate) and the stored ones (right of the plate). In the second row, these are P.putida WT (left of the plate) and the conjugates (right of the plate). In the last row, these are the two E.coli helper strain.

Cetrimide is a nitrogenous antiseptic molecule acting as an antibacterial. However, Pseudomonas bacteria are known to be resistant to this type of nitrogenous antiseptic, so cetrimide plates can be used for species-specific selection. We grew all the strains we had in the laboratory on cetrimide plates, with or without gentamicin, to check that helper and donor E. coli were not growing, and to isolate correctly transformed clones. As expected, the wild-type (KT2440) grew on cetrimide without antibiotics and did not grow with gentamicin, and our modified strains (RL) grew in both cases (middle row, right and left boxes). Unsurprisingly, the helper strains (PTRII and HB101) did not grow on cetrimide plates (bottom line, right and left boxes). On the other hand, one of our DH5a cultures was contaminated by a transformed KT2440 strain, since the plates grown with DH5a contained bacteria that were supposed to be unable to grow on this medium. After re-cultivating this strain on new cetrimide plates (top row, right and left plates), we observed that the strains from our contaminated culture (Pb) could grow, but the original strains as received could not (WT), except on the right-hand dish, where E. coli DH5a grew slightly, but not in the presence of gentamicin. In addition, we noticed that the P. putida KT2440 bacterial pellets obtained after centrifugation showed a reddish pellet, unlike the E. coli with a whitish pellet (not shown here). This test served as a further argument that the contamination was due to P. putida, since the pellets of the (Pb) strains were red, but not the DH5a from the original tube. This setback nevertheless taught us to handle the bacteria with much greater care, given its mobility, and the problem never recurred thereafter. From these cultures, we isolated 4 gentamicin-resistant transformed KT2440 strains for subsequent rhamnolipid production tests.

Achieved to have a mini-TN7 in the genome are not the goal of our technique and then, we had to test the rhamnolipid production.

Choi, KH., Schweizer, H. mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa. Nat Protoc 1, 153–161 (2006). https://doi.org/10.1038/nprot.2006.24.

* Hemolytic test:

In the course of our research, we discovered that biosurfactants such as rhamnolipids can have a hemolytic action. We tried to detect the production of rhamnolipids by highlighting this hemolytic capacity. To do this, we inoculated our transformed Pseudomonas putida on sheep blood agar plates, hoping that the rhamnolipids would degrade the blood through their hemolytic action and form a halo around the colonies. However, when we tried this technique, no halo was visible, indicating that rhamnolipid production was not effective or that this technique is not suitable for rhamnolipid detection. We repeated these experiments three times and no halo was ever observed, even after several days.

![](https://static.igem.wiki/teams/4643/wiki/bloodagar.png)

**Figure 2:** Culture of Pseudomonas putida KT2440 strains on sheep blood agar (7.5%) after 5 days at 30°C: from left to right, WT strain, strain modified to produce rhamnolipids RL1-RL4 and strain RL1 but in culture without glucose.

* Coomassie blue dye :

![](https://static.igem.wiki/teams/4643/wiki/dosage-controle.png)

![](https://static.igem.wiki/teams/4643/wiki/dosagerhamno.jpg)

**Figure 3:** Right: control curve obtained from an overnight culture of a wild type strain which does not produce rhamnolipids. Left: superimposed curves resulting from the analysis of the supernatant of all overnight cultures of rhamno-producing strains (in black RL1, in red RL2 and in blue RL3).

The methylene blue assay method is a quick and simple method for analyzing rhamnolipids by studying the complexation of rhamnolipids and methylene blue. The method is based on measuring the absorbance (at 638 nm) of the rhamnolipid-methylene blue complex which is distributed in a chloroform phase. Following the extraction of extracellular lipids with the chloroform phase, we produced absorption spectra between 200-700 nm of this chloroform phase. We emphasize that in the article on which we based ourselves, they obtain the same spectra shapes, leading us to believe that the experiment was correctly carried out. The first thing we observe is the absorption peak at 638nm which therefore hypothetically corresponds to rhamnolipids. On the other hand, this absorption peak is present for the same absorbance (approximately less than 0.500) both in the control (left) and for the strains supposed to produce rhamnolipids (right), which leads us to believe that there is clearly no production of rhamnolipid, and that this absorbance is perhaps due to the imperfect sensitivity of the method which could detect other types of extracellular lipids. We specify that we repeated these experiments three times, in particular by varying the glucose concentration, and obtained similar results.

Pinzon, N.M., Ju, LK. Analysis of rhamnolipid biosurfactants by methylene blue complexation. Appl Microbiol Biotechnol 82, 975–981 (2009). https://doi.org/10.1007/s00253-009-1896-9.

# Sophorolipids synthesis :

* Transformation :

Gibson assembly is a molecular biology technique used to assemble several DNA fragments into a larger DNA molecule. It is a powerful and efficient method for creating recombinant DNA constructs, such as plasmids, synthetic genes or other DNA constructs for genetic engineering or molecular biology research. Gibson assembly offers several advantages, including its ability to join DNA fragments without the use of restriction enzymes or ligases, and its capacity to seamlessly combine multiple fragments with overlapping sequences. This is why we decided to use this technique to create our plasmid.
We discovered that 4 enzymes were required for sophorolipid synthesis:
Cytochrome P450 CYP52 M1,
UDP-glucosyltransferase A1 (UGTA1),
UDP-glucosyltransferase B1 (UGTB1) and
Lactonase (or Lactone esterase)
These four enzymes are naturally produced in eukaryotic cells such as Starmerella bombicola. Our aim was to engineer P. putida to induce the synthesis of these four proteins, leading to the production of sophorolipids.
To this end, we turned to Pseudomonas expert Dr Victor Delorenzo. After several meetings with him, we set about designing a plasmid incorporating the genes for these various enzymes.
To create this plasmid, we asked Dr De Lorenzo to supply us with plasmid pSEVA438, which would serve as our backbone.
We also designed different biobricks containing each gene required for sophorolipid synthesis. First, we attempted to linearize pSEVA438 by PCR. After numerous attempts under different conditions, we were unable to linearize the plasmid using this technique (see photo of gels).
We therefore decided to linearize the plasmid with restriction enzymes.
Next, we used Gibson's assembly protocol to fuse our different fragments and create our plasmid for sophorolipid production.
Finally and after many trials, we successfully transformed Pseudomonas putida by electroporation. We also tried heat shock, and found that this technique resulted in less bacterial death.

![](https://static.igem.wiki/teams/4643/wiki/transfosophorolipids1.jpg)

**Figure 4 :** Bacteria shown here are grown in LB in the presence of streptomycin 30mg/L: WT KT2440 (left tube), transformed bacteria from electroporation (middle tube and right Erlenmeyer flask).

* **- SDS-Page and Western-Blot:**

After bacterial transformation, we wanted to check whether all four proteins were expressed. To do this, we analyzed protein expression in SDS-page electrophoresis and transformed pseudomonas after inoculation with 0mM, 1mM, and 2mM salicylate for 4 hours.
We stained our gel with Coomassie stain and observed different profiles between SDS-page and transformed pseudomonas. The appearance of bands between 50 and 75 kDa for transformed Pseudomonas could demonstrate the presence of new transformation-related proteins. Indeed, the WT strains, which were grown separately for 4H, all three (Figure ?, the three left-hand columns) show the same migration profile, indicating that the results are robust and reproducible. Similarly, the transformed strains have exactly the same migration profile. On the other hand, the profiles vary between strains, with some bands being highly concentrated in the wild-type but not in the transformed strains, and vice versa. Most interesting are the four bands located between 75 and 50 kDa in the profile of the transformed bacteria, as they correspond approximately to the molecular weight of the proteins contained in the sophorolipid plasmid (CYP52 61.8 kDa, UGTA1 50.5 kDa, UGTB1 46.1 kDa and lactone esterase 45.6 kDa). It should also be noted that our bacteria carry TAGs of a few kDa (less than 5kDa), which could vary migration slightly. Nevertheless, for the profile of transformed bacteria, there was no difference in the concentration of these bands in relation to the concentration of salicylate inducer. To explain this lack of induction, we may hypothesize that our promoter has a fairly strong basal activity (the XylS/Pm promoter being activatable with many different molecules (Morgenroth et al.) and that salicylate did not activate the promoter in any measurable way, or else that the plasmid is not functioning.
These results are encouraging, but we need to go further to determine whether the bands correspond to our four proteins. We had to design gene sequences with tags to easily target our neo-proteins. We didn't have time to carry out the Western blot for the wikifreeze but will do so before the Grand Jambore.

![](https://static.igem.wiki/teams/4643/wiki/sophorolipidssds.png)

**Figure 5:** Result of Coomassie blue staining of SDS-Page carried out on six conditions, three control conditions with a gradual induction of "no induction," "1mM salicylate," and "2mM salicylate" and three conditions transformed with the plasmid sophorolipid with the same gradual induction. Left well: BioRad dual color ladder.

# The future experiments :

* Sophorolipds dosage :

In our upcoming experiment, we will be utilizing High-Performance Liquid Chromatography (HPLC) to quantify the levels of sophorolipides in our samples. HPLC is a powerful analytical technique that is frequently used in the separation and quantification of complex mixtures. It will allow us to determine the concentration of sophorolipides produced by our modified Pseudomonas putida strains, giving us a precise measurement of our biosurfactant yields. This data will be crucial in assessing the effectiveness of our genetic modifications and in refining our strategies for enhancing sophorolipide production.

* Rhamnolipds dosage :

To determine the production of rhamnolipids, we will perform a rhamnolipid assay. This will involve culturing our genetically modified Pseudomonas putida strains and extracting the rhamnolipids from the culture medium. We will then employ a rhamnolipid assay kit to quantify the rhamnolipid concentration in the samples. This will offer us a quantitative measurement of rhamnolipid production, aiding us in assessing the performance of our engineered strains. Additionally, we will investigate whether modifying various culture conditions can further enhance rhamnolipid production.

* Combined Biosurfactant Production :

Our overarching goal is to create strains of Pseudomonas putida that are capable of producing both sophorolipides and rhamnolipides. This dual biosurfactant production has the potential to offer a broader range of applications and benefits, such as enhanced oil recovery and improved bioremediation. We will explore the feasibility of engineering strains that can simultaneously produce both biosurfactants by integrating the necessary genetic components for both pathways. This will be a challenging but rewarding endeavor, as it has the potential to open up new opportunities in the field of biotechnology.

* Advanced Characterization :

In our future experiments, we will dive deeper into the characterization of the sophorolipides and rhamnolipides produced by our modified strains. This includes analyzing their surface-active properties, emulsification capabilities, and stability under various environmental conditions. Such in-depth characterization is essential for understanding the potential applications and performance of these biosurfactants in different industrial and environmental settings.

* Application Development :

Our ultimate aim is to harness the biosurfactants produced by our engineered Pseudomonas putida strains for real-world applications. We will explore the use of sophorolipides and rhamnolipides in various fields, including bioremediation, enhanced oil recovery, and pharmaceuticals. Through collaborative efforts with industry partners and research organizations, we will work towards developing practical applications that can benefit society and the environment.