During our project, we fully experienced the concept of the engineering cycle. In order to make our design and the entire SeaCare project evolve, we tested each step of our thinking and analyzing our results was necessary to rethink our first idea. Here, we will describe an example of how we worked with this concept. Indeed, it was especially applied during the search for a molecule adherent to the microplastics of interest.

Peptide (engineering cycle 1)

Design
The idea of working with those peptides comes from the fact that they were used in some patents that shows their ability to adhere to PE and PP plastics. These peptides are all about 1,32 kDa. We decided to do the experiments with all of their peptides (7 PE and 7 PP). To clone those peptides, we decided to link them with a linker which is also linked to GFP. For that, we use EcoRI and HindIII as restriction enzymes (Figure 1).

Production
1- The 14 peptides used were found in patents where they are known for their supposed adherence to our 3 main plastics (polyethylene, polypropylene and polystyrene).
2- We used an E. coli strain (BL21) and a plasmid JS 321 (for the GFP).
3- An amplification of our inserts has been done by PCR.
4- Digestion and ligation: digestion by restriction enzymes EcoRI and HindIII were performed in order to access the plasmid and ligation was performed in order to connect the inserts to the plasmid.
5- A transformation in DH5 alpha was carried out in order to introduce the peptide genes, and thus transform the plasmid.
6- We verified our cloning results by doing a PCR screen.
7- A transformation in BL21 was done in order to be able to express proteins subsequently.
8- Then we had to purify our peptide after the induction; inoculation and centrifugation of E. coli.

Testing
To test them we imagined various protocols and contraptions but we settled on the following one. As the pilot test seems to work, we harmonized the concentrations at 350 ng/µL and prepared a 500µL solution for each peptide with a high concentration.
We then put 50 µL of the peptide solution on each plastic (polystyrene and polyethylene), testing both the peptide on its dedicated plastic but also on the other one, to verify to what extent it’s specific. Then, we used a Tecan : A Dedicated Fluorescence Reader; and used the SDS as a "white" when testing the plastic.
To analyze the results, we calculated from the OD504 we had at the beginning, every protein loss (on the washes, and the liquide we retrieved) by subtracting them. We replicated those steps to create a "replica" which verifies the quality and reproducibility of the protocol.

Analysis
All peptides adhere differently to plastic and their specificity of adherence also seems to be different. While all the PE peptides seem to specifically adhere to PE plastic, the results are the opposite for PP peptides which are not specific. Our experience shows that all the PE peptides that we were able to test all adhere to PE plastic. On the other hand, some PP plastics don't adhere to PP plastics while some do.

Phage display (engineering cycle 2)

Design
Given the previous results our objective is to find a molecule with a high affinity for polyethylene, polypropylene and polystyrene that is much bigger and much more specific to the type of plastic it adheres. Therefore, we thought of VHH antibodies. A VHH antibody (or nanobody) is the antigen binding fragment of heavy chain only antibodies. They are very specific and their molecular weight is approximately 15KDa. We decided to do this experiment for polyethylene and polystyrene only because of the complexity of the experiment.

Production
1. The phage library was builded by a scientific research team by merging a VHH to a phage coat protein gene (here the pIII), causing the phage (here the M13) to "display" the VHH.
2. Binding: once exposed to the plastic, only a few phages interacted with targets in these libraries.
3. Washing: phages that are not bound can be washed away, leaving only those that have an affinity for the plastic.
4. Elution: the target-bound phage is recovered through elution.
5. Amplification: Eluted phages that exhibit specificity are used for direct bacterial infection and amplification of the recovered phage.
The cycle is then repeated two to three times to select the best binding sequence in steps.

Testing
For each plastic (polystyrene and polyethylene) we screened 21 VHHs by ELISA. For this, we used a primary antibody anti-HA which will recognise the HA tag in the VHHs. We then used a secondary antibody anti-mouse-HRP which is conjugated to the Horseradish Peroxidase enzyme acting on various chromogenic substrates to produce many different colors. We used the BM Blue POD substrate and allowed us to detect a reaction by reading the optical density at 450 nm. This experiment was done with plastic as a target (polyethylene or polystyrene) and without the plastics (PBS).

Analysis
For polystyrene, we had several positive results compared to the positive and negative control of the ELISA but none of them seemed to have a very specific affinity for plastic because the PBS control was positive as well. We therefore choose 2 VHHs (in yellow) whose ELISA reaction was positive, therefore with an affinity for polystyrene but also for other surfaces. We named this 2 VHH PS-VHH1 and PS-VHH2 respectively. For polyethylene, surprisingly, 2 VHHs seemed to have a specific affinity. In fact, we can see (in green) that the ELISA was positive for these VHHs when the target was polyethylene, and that the ELISA was negative when we used PBS. Therefore, we think that these VHHs are specific for polyethylene. In addition, their multiple alignment and their predicted three dimensional structure suggest that we did in fact produce 4 VHHs with 3 CDRs (Complementarity Determining Regions) responsible for antigen recognition and thus antibody diversity. However, it would be interesting to produce these VHH.

Future experiments
It would be interesting to produce these PE-VHH, purify them and test them to verify their specific affinity for plastic. If there are not, it would be interesting to redesign the phage library and the panning protocol. If they are specific, it would be interesting to produce specific VHHs for polystyrene and polypropylene by adapting the protocol and the laboratory material.