Difference between revisions of "Team:Toulouse-INSA-UPS/Demonstrate"

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                 Our second construction consisted on our protein Cerberus but lacking the un natural 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 <i>in vivo</i> and <i>in vitro</i>, 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 (figure3.a), biotinylated <i>in vivo</i> in <i>E. coli</i> and Scygonadin, an anti microbial peptide, produced in <i>Pichia pastoris</i> (figure 3.b).
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                 Our second construction consisted on our Cerberus protein but lacking the un natural 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 <i>in vivo</i> and <i>in vitro</i>, 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 (figure3.a), biotinylated <i>in vivo</i> in <i>E. coli</i> and Scygonadin, an anti microbial peptide, produced in <i>Pichia pastoris</i> (figure 3.b).
 
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Revision as of 12:34, 14 October 2018

DEMONSTRATE

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) azido-L-phenylalanine moiety. We also successfully set up a workflow allowing the synthesis of Cerberus (Figure 1).

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

Validation of the Three Binding Heads

Cellulose Binding

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.

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

Biotinylated compound affinity

Our second construction consisted on our Cerberus protein but lacking the un natural 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 (figure3.a), biotinylated in vivo in E. coli and Scygonadin, an anti microbial peptide, produced in Pichia pastoris (figure 3.b).

Fluorescence Retained
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

Our plateform aims to have a binding capacity as wide as possible. The azide groupment offer a high efficiency covalent bound formation through cycloadditionreaction, 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.

Fluorescence Retained
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

Production and Functionalization of Bacterial Cellulose

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Figure 1: Bacterial cellulose production

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

Conclusion

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

INSA Toulouse UPS LISBP TWB Defi Adisseo Foundation ESF Catalyses


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