Engineering
The Engineering process is composed of four stages: Learn, Design, Build, and Test
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.
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...
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:
To create this plasmid, we requested Dr. De Lorenzo
to provide us with the pSEVA438 plasmid, which will serve as our backbone:
Then we designed the 4 bricks, each containing a gene:
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.
Testing Rhamnolipid Production / Insertion of Rhamnolipid Genes
1. Antibiotic Selection
Pseudomonas putida was initially the only strain expected to grow on cetrimide agar.
However, we discovered that E. coli DH5α wild type (WT) could also thrive in this medium.
The P. putida colonies exhibit distinct characteristics compared to the DH5α 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
DH5α WT and P. putida WT, but the conjugating P. putida strain remains viable.
Upon further investigation, we encountered a tube of E. coli DH5α that could grow on cetrimide
with gentamycin. However, a colony comparison revealed that it was indeed P. putida that had contaminated
this E. coli DH5α 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:
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
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:
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:
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.