|BBa_K2668010||CERBERUS: binding of biotinylated compounds, binding to cellulose, can covalently bind alkyne decorated molecules||basic||working|
|BBa_K2668020||SIRIUS: strong binding to cellulose||basic||working|
|BBa_K2668070||BFP-Avi-tag: fluorescence in the blue wavelength, can be biotinylated||basic||working|
Parts used on the project but not deposited on the registry
This part results in the fusion of scygonadin, an antimicrobial peptide from the mudcrab Scylla paramamosain, with an Avi-tag, a short peptidic sequence recognized by BirA in order to biotinylate proteins in vivo. It was used to prove the affinity of Cerberus to biotinylated compounds and to check if they keep their biological activity thereafter. Scygonadin was planed to be produced in Pichia pastoris since it is an eukaryotic, microorganism, the production of an antibacterial peptide should not harm it.
This part was used as a negative control for the affinity of Sirius towards cellulose. Its production was achieved under the exact same conditions as Sirius. The designed part was cloned into pET28 which includes an His-tag and can therefore allow us to purify it using cobalt column. It is always a pleasure to produce red proteins as it is very beautiful!
BirA is the E. coli biotin ligase and has a high affinity for Avi-tag, a short peptidic sequence. It was co-expressed with mTagBFP and scygonadin for in vivo biotinylation. We used the pETDuet-1 for its production in E. coli. We got inspired by this system for the design of a double protein expression plasmid in Pichia pastoris for that we used the pPICZalpha to clone BirA into it and cloned the function to be biotinylated into the pGAPZalpha. Then we cloned the pGAP expression cassette (containing the CDS fused to an Avi-tag, a promoter and a terminator) .
Cerberus was initially designed to use the classic tetrameric streptavidin, but we chose to also design a monomeric version using an engineered version. It was a good choice because the tetrameric version did not work...
No dogs were harmed over the course of this iGEM project.
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