Project Description

Problem

This year, we are interested in the problem of pollution of the Mediterranean Sea by microplastics. Indeed, the Mediterranean Sea is one of the most polluted seas in the world. To date, the surface waters of the Mediterranean Sea contain 84,800 microplastics per km2, and this figure increases every year. However, these data only concern surface pollution due to the lack of studies in deep waters. Scientists therefore expect the level of microplastics to be much higher when the entire water column is taken into account. The presence of microplastics in the sea is not only a problem for all marine biodiversity but also for humans. Indeed, recently, a scientific research team found for the first time microplastics in human blood. In view of the threats posed by microplastics in the Mediterranean Sea to us and to the marine ecosystem, urgent action is needed.

Our solution

Our project, of global and local scope, is therefore part of a desire to degrade the most abundant microplastics in this sea, namely polyethylene, polystyrene and polypropylene found in our packaging, everyday waste. This process would reduce the amount of microplastics present and thus protect the marine ecosystem from human pollution. Our main objective is to use synthetic biology to produce a biological system capable, once introduced into the "chassis" bacteria, of detecting and adhering to polyethylene, polystyrene and polypropylene and to produce a structure that can also attach itself to microplastics called "plasticosome" (derived from cellulosome). This "plasticosome" can contain a large number of enzymes known to be capable of degrading the polymers of interest such as Laccase which in our project will constitute the proof of concept. Thus, this biological system could be used in a device capable of recovering microplastics from the Mediterranean Sea in order to degrade them. In parallel, a major objective of this project is to raise awareness of our problem among a diverse public, students, Marseilles and French people in general. Indeed, we all know that the massive use of plastics must be reduced and eventually stopped in France but also worldwide. France is the first producer of plastic waste in Europe and its recycling rate is lower than that of its European neighbors. Moreover, the concentration of plastic waste in the sea is particularly high near Marseille. This is why we wish to explain all the stakes of the massive use of plastics as well as to be able to sensitize the population on the management of waste by taking into account the sanitary, social and environmental aspects.

Our project is divided three objectives :



Figure 1 : Bacterial adhesion on microplastics

Figure 2 : Secretion of plasticosome by bacteria

Figure 3 : Plasticosome adhesion on microplastics

Figure 4 : Degradation of microplactics by enzymes

Phage display

Finding a bigger and more specific molecule by phage display
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 to than the peptides found in the litterature.

Therefore, we thought of VHH antibodies. A VHH antibody (or nanobody) is the antigen binding fragment of heavy chain only antibodies.They were discovered 25 years ago and they are naturally produced by camelids and sharks. The main advantages of VHH is that they have exceptional binding properties, they are not too big or too small (approximately 15 KDa) and they are very stable (Bever et al., 2016).

Our laboratory was located in the same building as a team specializing in VHH and working with these molecules every day (the host-pathogen interaction team directed by Alain Roussel at the LISM in the CNRS of Marseille) which allowed us to use one of their Phage’s library. This library is called Loupio, and was created from the immunization of a llama with a protein X (which its name cannot be released) with a diversity of 5.1x10^6. We would like to clarify that this phage bank was not created by our iGEM team, it was given to us by this specialized team at the LISM which has been generating VHH since 2011 and they assured us that the immunization of the llamas is done in partnership with an approved farm and the processing of the blood follows a very precise organization.

This phage’s library was used to follow a protocol of the phage display technique that we adapted in order to find the VHH’s with high affinity for polyethylene and polystyrene. Polypropylene (one of the plastics that we were interested in for this project) was not used in this experience because as we were developing and adapting our phage display protocol, we discovered that a majority of the material used in the laboratory was made of polypropylene and therefore not adapted for our protocol. We made the decision that we will start with polyethylene and polystyrene and then if our protocol works, we will adapt our laboratory material in order to find VHH specific to polypropylene ; unfortunately we did not have enough time.

Phage display technology generally consists of 5 steps : (Bazan et al., 2012)
1. Create the phage library : A gene encoding the VHH of interest is merged with 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 will interact 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.

Figure 1 : The phage display technique principle (The Overview of Phage display)


References

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