Compounds Fuctionalization

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Fluorescence

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Once our system proved, we tried to associate several molecules in order to prove the system versatility. We successfully associate fluorescein to the streptavidin head. We made it possible thanks to the click chemistry with which we brought the necessary biotin for the streptavidin interaction.

Color

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Thanks to Sirius and its brightness, we could observe how the color remained on Bacterial Cellulose (figure 2) even after dozen of washes!

Antibacterial Activity

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We also conjugated an antimicrobial peptide, scygonadin, to the streptavidin, even if we need to optimize protocol (figure 3) we are comfident in the remaining biological activity after peptide binding on Cerberus.

Paramagnetism

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For the AzF head, we decided to bind an inorganic molecule: paramagnetic beads, and it was a success (see the video below, figure 4). So our Cerberus platform has allowed us to make magnetic cellulose.

Versatility of the system

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All of our experiments demonstrated the versatility of our system. Indeed, click chemistry is a real advantage to increase number of possibilities of functionalities. DBCO-biotin compound may be used as an adaptor between the streptavidin head and the AzF head as showed in figure 6.

Perspectives and conclusion

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We successfully set up a workflow allowing the synthesis of Cerberus, a proteic platform that is composed of a cellulose-binding molecular module (CBM3a) fused at its N-terminal part to a biotinylated molecule-binding head (monomeric streptavidin) and at its C-terminal part to an unnatural amino acid (UnAA) 4-azido-L-phenylalanine moiety. Combining molecular modeling, synthetic biology and chemistry, we proved that Cerberus is a valuable and versatile platform allowing to functionalize cellulose with organic and inorganic compounds.

We can also plan to associate carbon nanotubes and graphene to AzF head in the aim to change cellulose fibre rigidity and create conductive cellulose, respectively. In order to do that, we successfully activated these compounds by diazotization reaction. The click chemistry assay and validation protocol need improvement to obtain our goal.

Bacterial cellulose has unique properties such as its natural purity may give it a bright future in the industries, that is why we decided to produce functionalized bacterial cellulose in vivo in reactor. As a first step towards the production of functionalized bacterial cellulose in a reactor, we demonstrate that CBM3a module of Cerberus fused to Red fluorescent protein is able to make bacterial cellulose fluorescent. Moreover, our first production of Orthos in Pichia pastoris are very conclusive, but we have to optimize the production.

We truly feel that Cerberus platform is a very convenient and efficient solution to facilitate molecular work for iGEM projects and laboratory work in general. In fact, we can combine the use of two binding sites to purify simply molecules of interest (cellulose/streptavidin or cellulose/AzF or streptavidin/AzF). For example, we can imagine purify cells via biotinylated membrane proteins. An addition of Cerberus protein can permit the interaction between the monomeric streptavidin of Cerberus and the biotin of protein. Then, two possibilities are open to us to purify using either cellulose or paramagnetic beads. After attraction, we can release molecule of interest by using the TEV protease cleavage.

Finally, we can also imagine to change our CBM3a to another proteic domain to associate functionalizing proteins to new materials as plastic to facilitate its degradation for example.