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Our Problematic(s!)

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A specificity of our project is that we did not start its construction from a single problematic but we actually started from a multiplicity of problems. For example:

• How to produce band aids with antibiotics?
• How to produce paper on which we can print electronic circuit boards?
• How to create new fluorescent fabrics?
• How to create packaging exhaling the smell of the food inside?
• How to manufacture feminine protection preventing toxic shock syndrom?

and so on

This forced us not to think about a specific real world problem but to tackle a technical lock common to all these projects realisations: functionalising cellulose.

Each question could have been addressed using a dedicated physico-chemical or even synthetic biology solutions. However, we considered this an awkward way to solve such a generic problem and we decided to search for a generic and more elegant solution. This is how Cerberus has been brought to life: instead of one construction for each question, we brainstormed creating a solution for all the questions in depth.

In the specifications, the most important features we sought for were genericity and versatility. We found rapidly what molecules should be grafted to cellulose, but they were of a very different nature to be associated through a common property: graphene, antibiotics, carbon nano-tubes, fluorescent proteins or chemicals, paramagnetic beads, etc.

Emergence of the Cerberus Platform

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It soon appeared that there were three main properties important for us: (i) to bind cellulose, (ii) to bind organic compounds, (iii) to bind inorganic molecules. This is how we designed a three-headed protein where each head responds to one of these specific questions (and how we chose Cerberus as a name for it!).

The cellulose head was the easiest to design. Cellulose Binding Domains have already been successfully used by fellow iGEMers (for example, Imperial 2014 or INSA Lyon 2016). This required a little sequence optimization but it was easy.

The next head was the streptavidin domain. Here we had to choose between the classic tetrameric streptavidin and a monomeric streptavidin unsuccessfully used by iGEM UGent Belgium 2016. We tried both. We also added a TEV protease site to add the possibility of releasing the molecule associated with streptavidin.

The last head was the trickiest. To bind non organic molecules to a protein, we had to search in state-of-the-art synthetic biology concepts, at the interface with chemistry. We ended up with choosing unnatural amino acid incorporation in the Cerberus platform and the use of click chemistry to create covalent bond between inorganic or semi-inorganic compounds and our protein.

Advantages of the Cerberus Design

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We could have created a specific protein for each of the applications. However, this would have required analyzing the stability and feasibility of each of them, independently. In contrast, Cerberus is designed with strong stability of its heads but flexibility of the linkers to associate them, which allow compatibility even with complex molecules such as graphene. Besides, there are endless possibilities to modulate the Cerberus structure, for example by replacing one head by another or changing the CBM3a domain by a different domain with another type of affinity. DBCO-DBCO or DBCO-Biotin compounds could also allow the linkage of Cerberus-associated molecules together. So in our opinion, Cerberus could be the basis of many other developments, well beyond cellulose functionalisation.

Ensuring the Project Success

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All iGEM teams have great ideas and concepts, but the competition is a big challenge in a very short period of time. We were lucky to succeed in demonstrating the Cerberus efficiency (see Demonstrate), but this is not only about luck. Here are the reasons which had improved our chances to do well.

1. Modelling. Cerberus was trickier to design than we had expected. We had no idea about the reactivity and conformational problem that could issue from the unnatural amino acid. The Cerberus linkers that bind the domains altogether could have easily destabilized the whole protein too. Modelling was therefore a great way to assess in silico that we were going in the right direction (see the Model section ).
2. Human Practices. These were central for us. Downstream of the project, the interaction with the public and society reassured us of the importance of what we were trying to achieve. Upstream of the project, the IHP helped us a lot for the project fine tuning and feasibility (see the Human Practices section).
3. Division of the project in modules. This is classically done by iGEM teams and this is indeed a crucial point to ensure that the whole project is not stuck because of one of its aspects (see the Design section).
4. Multiplying the strategies. We chose for example to try both tetrameric and monomeric streptavidin. Likewise, we tried both production by E. coli and by the yeast P. pastoris. So yes, this is extra work to do, but finally, tetrameric streptavidin never worked in our hands, and P. pastoris production was disappointing too. Therefore, we could have easily failed the whole project without this multiplication of strategies (see the Results section).
5. Multiplying the assays. We had choices to make about what functions we wanted to graft on cellulose. We tried several of them. Some succeeded rapidly (fluorescence, magnetism), some still need improvements (antibiotic), and some took too long to be completed (graphene, carbon nanotubes). Multiplying the assays improved our chances to succeed in a very short and required schedule (see the Demonstrate section ).

Rupture Technology and Lifecycle

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We thoroughly investigated the many consequences of Cerberus for the environment, the manufacturer and the end user’s everyday life through our ethical analysis (see IHP section) and Entrepreneurship efforts (see Entrepreneurship section). These were essential but difficult exercises since the possibilities and applications issued from Cerberus are so large and hard to anticipate. Nevertheless, it reassured us in both the innovations made possible by Cerberus and the improvement it will be for groundbreaking processes. It opens up so many possibilities that everyone is excited about what it could bring to each of us in a future which is now envisigable.