Adding new properties to cellulose

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Color

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It was not initially planned to add colors to cellulose. However, mRFP1 has the property to provide a red hue without excitation. This was easily observable on bacterial cellulose after incubation with CBM3a-mRFP1 (Figure 1). This could be of special interest since a lot of proteic dyes are available (see iGEM Imperial 2016). The current dyes used in the textile industry are often toxic to people or the environment. Cerberus association with proteic dyes could thus be a safer alternative solution.

Fluorescence

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The first sought-after property of mRFP1 is for sure fluorescence. We successfuly produced fluorescent cellulose by combining cellulose, our platform and fluorescent dyes in several ways : by adding biotinylated fluorescent compounds, by clicking fluorescent molecules with an alkyne function or even by testing the CBM3a-mRFP1 construction on bacterial cellulose (Figure 2). This proves that physico-chemical properties of protein could be transfered to cellulose and allow to imagine many other developments (luminescence for instance).

Antibiotic

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We also conjugated an antimicrobial peptide, scygonadin, to the streptavidin. The first results seemed to indicate this could work (Figure 3) but it was not as a definitive result as the other functionalization assays we have performed. This will require further testings since such a functionalization arouse very important expectations from the public (see Human Practices).

Paramagnetism

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Last but not least, we validated the grafting of paramagnetic beads to cellulose (Figure 4). So, our Cerberus platform has allowed us to create paramagnetic cellulose. In our opinion, this is the functionalization that should permit the largest panel of applications, especially in combination with other functions as Cerberus allows. Just imagine all the application in purification of compounds or cells!

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.

A workflow to produce functionalized cellulose

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This project was not only about functionalizing cellulose but ambitioned to define the whole process from component purification to production in vivo (Figure 1).

Indeed, we have defined and validated the basis to model our platform. This allows testing in silico any further modifications that could be of interest for Cerberus. Next, we demonstrated the efficiency of the Cerberus production in E. coli. This validated our choices for the strain, vector and culture conditions (IPTG, AzF, arabinose…). Then, we also successfully purified Cerberus and shown this through SDS-PAGE and Western Blot. As previously described, production of biotinylated compounds was validated as well as cellulose functionalization using Cerberus. We went even further and started the production of cellulose in vivo with Gluconacetobacter hansenii. The next step would have been to demonstrate the possibility to co-culture this strain and a strain synthesizing Cerberus and the function. Then, the final stage will be to start the production of cellulose in vivo. Even if we did not have enough time to each this final step, it was thought from the beginning of the project, as it was for this whole workflow.