First of all, our project comes from the realization of a problem that affects our local coasts: green tides. News media, global as well as local journals report a recurrent problem with economical and social issues. Deeper investigation reveals that the hazardous phenomenon associated with green tides is due to the apparition of anoxia zones, which induces the switch of bacteria (especially sulfate-reducing bacteria or SRBs) into anoxic metabolism [1]. This leads to the emanation of hydrogen sulfide (H₂S) by the SRBs, which is a dangerous gas.
Two approaches were then proposed :
I) target the green tide phenomena
or
II) target the H2S emanation
Further researches, by consulting scientific resources and data, has allowed us to identify a specificity of these green algae, that could be used as our target: ulvan. It’s the specific polysaccharide and main component of the Ulva cell wall and precursor of H₂S production. The publication [2] on which we based our project presents the degradation of the cell wall by an enzyme group.
Imagine
The first step of our project consisted of selecting 3 enzymes to reduce the algal mass. Our first purpose was to pulverize our enzymatic cocktail directly on stranded algae to decompose them. This project was presented to algae experts to review it, especially to Mrs. Justine Dumay who shed light on ethical and environmental issues with this project. Instead of just degrading the blooms, she suggested valorizing the algae after their collection. Thus, our team began to think about different ways to valorize the algae. Soon, we have oriented our research to optimize H₂S production, which is an economically interesting compound because it can be transformed into sulfuric acid (H₂SO₄), a widely used compound in the chemical industry, especially as a strong acid.
Our scientific method : The DBTL Workflow
As a scientific method, we use the DBTL workflow which can be represented as a cycle. This engineering method is meant to improve our circuit by following a rigorous approach.
To reach our goal of transforming H₂S into H₂SO₄, we developed a brand new project based on our initial one, and this project was what would become the A3 project. The new objective was no longer to solve the problem of green tides but to exploit it. The dangerous gas which was the reason why green tides were so problematic was going to become our strength.
To do this, we decided to stimulate rather than prevent the production of H₂S in the laboratory by introducing a new type of enzymes (in addition to the degradation enzymes) that would allow better access and release of the sulfates during the ulvan degradation. These enzymes are sulfatases and will work in a tandem with bacteria that are present in the natural environment of our algae. Some of the bacteria already present with the algae are called SRBs and are able to use the sulfated sugar of the algae’s cell wall to produce H₂S in anoxic conditions.
Build
The H₂S production method consists of expressing our enzymes in several plasmids (one enzyme of interest per plasmid) that are transfected in an E. coli chassis. The E. coli BL21 (DE3) strain has been chosen and is widely described as an effective transfected strain.
We selected our enzymes based on the article written by Reisky et al. (2019) which describes the enzymatic cascade degradation of ulvan. Then, we studied our enzymes’ sequences on UniProt and NCBI. After this, we performed the design of our plasmid on SnapGene. We used the Gibson Cloning Assembly technique because this method is already used and well known by one of our supervisors.
For the plasmids, we based the design on the same article [2]. In this publication, they used a pET-type plasmid with an IPTG inducible promoter for the 6 enzymes we have chosen (degradation enzymes and sulfatases) and a plasmid with arabinose inducible promoter for the FGE enzyme.
Test
First test :
It consists in the verification of the action of the enzymes produced by our E. coli BL21 strain
Principle of the assay: Test of an enzyme cocktail that breaks glycosaminoglycans glycosidic bonds.
The products of enzymatic degradation will be specific to our chosen enzymes.
Electrophoresis allows us to identify the migration of the different disaccharides in several bands.
→ Sugar quantification by fluorescence
→ Informs on the enzyme's action
Second test :
It consists of the verification of the sulfatases action and free sulfate production.
HPAEC-PAD :
Principle of the assay: Separation of the different sugar by anionic chromatography. Then, we run an analysis of the separated sugars by the PAD detection cell.
→Sugar quantification
→Informs on the sulfatase’s action
Third test:
It consists of the verification of the presence of SO42- ions, by a chemical reaction.
Precipitation with BaCl2 :
Principle of the assay: Identification of SO42- ions by the apparition of a white precipitate of BaSO4.
→SO42- identification
→Confirms the correct action of enzymes and sulfatases
If the amounts of SO42- - are still too low or null compared to the negative control, these solutions could be considered :
- Changing for enzymes with similar action
- Trying other measurement techniques
Learn
- Does it work? If it doesn't, how can it be improved?
This step consists of conducting troubleshooting our circuit. According to the different information at our disposition, we will proceed by elimination. We will test different conditions for the adjustable parameters (e.g to determine the most optimal temperature, we will repeat the tests with slightly different temperatures than the ones that give the best result). Many variables can be tweaked, and the nature of the issue will give us clues on what could be the cause. We will get back to that in the next part (“improve”).
- If it works, what results are expected?
We expect to detect a significant increase in the measurement of SO42- concentration. If it doesn’t work, there will be little to no change relative to the control.
Improve (redesign)
There can be many different reasons for dysfunction related to our genetic circuitry. Please see Table 1 for a non-exhaustive list of potential issues concerning our biological constructs and/or hardware and their possible solutio
Potential reasons for the lack of results
Potential causes (Learn)
Potential solutions (for a new cycle of Build-Test-Learn)
Lack of induced protein
Toxic proteins
Currently: E. coli BL21 (DE3) For the next DBTL cycle:
Use an E. coli pLysS strain
Use a new resistant strain (e.g. E. coli BL21-AI)
Low concentration of inductor proteins
Increase the concentration of Arabinose (if FGE synthesis is too low)
Increase the concentration of IPTG (if the synthesis of the other enzymes is too low)
Vary the environment (pH, temperature, buffer, etc.)
Test other enzymes with similar target
Presence of residual O2 in the tanks
Use nitrogen bubbling to eliminate O2
Enzymes accessibility issues
Density of algal mass is too high
Non- homogenous repartition of enzymes or reagents
High level of impurities (sand for example)
Regular stirring of the medium
Preliminary crushing of algal masses
Addition of a previous purification step for sand removal
Directed mutagenesis to increase the efficiency of the enzyme
Chromatography problem
Elution is not good enough
Eluant effectiveness is too low
Change the eluent
Increase eluent concentration
Increase the pH value
Table 1 Troubleshooting of several potential issues with the system and their respective possible solutions. This list is obviously non-exhaustive as a multitude of parameters have to be taken into account [3].
Sources
[1] Muyzer, G., & Stams, A. J. (2008). The ecology and biotechnology of sulphate-reducing bacteria. Nature reviews microbiology, 6(6), 441-454.
[2] Reisky, L., Prechoux, A., Zühlke, M. K., Bäumgen, M., Robb, C. S., Gerlach, N., ... & Song, T. (2019). A marine bacterial enzymatic cascade degrades the algal polysaccharide ulvan. Nature chemical biology, 15(8), 803-812.
[3] Brophy, J. A., & Voigt, C. A. (2014). Principles of genetic circuit design. Nature methods, 11(5), 508-520.
igemnantes2020@gmail.com