Team:Stanford-Brown-RISD/Improve

4xCBD Fusion Protein Expression System T7 promoter, a salI cut-site, then a start codon then a Flag, Lumio, 6x polyhistidine tag, followed by BBacillus circulans chitin binding domain, flexible linker, S. griseus chitin chitin binding domain, then another linker, followed by Bacillus circulans chitin binding domain, flexible linker, S. griseus chitin binding domain, stop codon, a bglII cut-site, and finally t7 terminator.

This composite part contains all the biological DNA machinery to constitutively express the fusion protein 4xCBD when functional T7 DNA polymerase is inside the cell with the plasmid carrying the composite part. The fusion protein is four chitin binding domains fused together with flexible linkers. Our rationale was that four chitin binding domains would allow each protein to bind strongly to chitin than a single chitin binding domain alone, and also allow for the linking of two different chitin surfaces when pressed together with this protein acting as a glue in between them. This is an improvement upon the basic part BBa_T2028, which is a single B. circulans chitin-binding domain. Our fusion protein includes two BBa_T2028 B.circulans chitin binding domains, and an additional two chitin binding domains from S. griseus. We diversified the chitin binding domains to allow for easier DNA printing of our part. This whole linked structure also incorporates a 6x polyhistidine tag for the purification of the protein product.

We were able to successfully express this construct in the BL21 derivative E. coli cell line T7 Express from New England Biolabs, purify this protein using Ni-NTA spin columns for his-tagged protein purification, and test it as a bioadhesive on mycelium (as the fungal cell wall incorporates chitin) and cardboard substrates. Although it actually bonded cardboard together slightly stronger than it bonded mycelium together, it was among the top of our tested bioadhesive proteins on mycelium! To read more, please see our wiki page linked here.