Team:Toulouse-INSA-UPS/Demonstrate

DEMONSTRATE

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The integration of our entrepreneurial and social analysis led us to consider finding a way to functionalize bacterial cellulose. For this purpose, we designed a three headed proteic platform that we named Cerberus in reference to the mythological dog. It 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. We also successfully set up a workflow allowing the synthesis of Cerberus (Figure 1).

Figure 1: Workflow of our Cerberus project experimental validation process.

• Validation of the System
• Functionalization
• Bacterial Cellulose
• Conclusion

Validation of the Three Binding Heads

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Cellulose Binding

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We designed a fusion protein between the Carbohydrate Binding Module type 3a and a fluorescent reporter and named it Sirius as a reference to the brightest star of the norther hemisphere. Its purpose is to validate the association of our system towards cellulose. We demonstrated, using mRFP1 alone as negative control, the binding of our protein as showned in Figure 2. We were then confident in the binding of our protein to cellulose.

Figure 2: Picture of cellulose pull-down assay with mRFP1 alone (left tube) or with the Sirius construction (right tube)

Biotinylated compound affinity

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Our second construction consisted on our Cerberus protein but lacking the unnatural amino acid. This construction aimed to prove the binding affinity of our system toward biotinylated compounds. As biotinylation can be both performed at high yield in vivo and in vitro, this construction is already a molecular binding plateform, allowing the purification of biotinylated compounds at low cost, using cellulose. We validated this construction using biotinylated mTag Blue Fluorescent Protein (Figure 3.a), biotinylated in vivo in E. coli and Scygonadin, an anti microbial peptide, produced in Pichia pastoris (Figure 3.b).

Figure 3: a) Functionalization of cellulose with Orthos bound to biotinylated BFP. Fluorescence remaining on cellulose fraction after several washes (*Mann Whitney test p-value 0.01) b) Halo of inhibition of Orthos alone ( C), scygonadin alone (S) and Orthos+scygonadin (C+S) samples

Click chemistry

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Our plateform aims to have a binding capacity as wide as possible. The azide groupment offers a high efficiency covalent bound formation through cycloaddition reaction, mostyl known by Click chemistry. The possibilities offered by this system are therefore included in the unnatural amino acid : 4-azido-L-phenylalanine. We designed our protein to bear an Amber stop codon for unnatural amino acid integration in our plateform. We confirmed the activity of this integration using DBCO-Fluorescein as showed in figure 4. We also functionnalized cellulose using paramagnetic beads (see video below) which shows the broad range functionnalization possibilities that our system offers.

Figure 4: Functionalization of cellulose with FITC conjugated to Cerberus. Fluorescence remaining in cellulose fraction after several washes (quadruplicate test). (*Mann Whitney test p-value 0.03)

Figure 5: Video of the Cerberus-functionalized paramagnetic cellulose: on the left, control with paramagnetic beads alone; on the right, Cerberus-paramagnetic beads.

Compounds Fuctionalization

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Production and Functionalization of Bacterial Cellulose

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Figure 1: Bacterial cellulose production

Figure 2: Revelation of functionalized cellulose with Sirius (right) and mRFP1 alone (left) on UV table

Conclusion

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No dogs were harmed over the course of this iGEM project.

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The whole Toulouse INSA-UPS team wants to thank our sponsors, especially:

And many more. For futher information about our sponsors, please consult our Sponsors page.

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