Part:BBa_K2868024
csgA:CBD Expression system
T7 promoter with RBS, followed by a SalI cut-site, then coding region for csgA, followed by a flexible linker, followed by the NEB chitin binding domain, which is followed by the multi-tag of Flag, lumio, and 6x polyhistidine, and a stop codon, then a BglII cut-site, and finally a T7 terminator.
This composite part is for the expression of the fusion protein we designed, csgA-CBD. Expression requires a T7 RNA polymerase because of the T7 constitutive promoter. It is a csgA curlin precursor of E.Coli coding region linked to a Bacillus circulans chitin binding domain with a 6x polyhistidine tag.
CsgA has well documented amyloid formation behavior and is a main component of E.Coli biofilms, so we theorized that if it were fused to a chitin binding domain the resulting fusion protein would exhibit exceptional adhesive properties to chitin while also remaining cohesive with other csgA-CBD proteins. These traits are extremely attractive for use as a biological adhesive on mycelium substrates, as the fungal cell wall is largely composed of chitin.
We were able to successfully express our fusion csgA-CBD protein using this composite part on a pSB1C3 plasmid backbone. We then purified the resulting csgA-CBD protein using standard his-tag purification protocols on Ni-NTA resin spin columns, followed by isoelectric precipitation. The final pure protein product tied for the strongest bonding strength on a mycelium substrate out of the proteins we tested, and had the best ratio of strength on mycelium to strength on cardboard. To read more about our design process, laboratory procedures, testing data, and results surrounding this part, please read our wiki page on the subject here: http://2018.igem.org/Team:Stanford-Brown-RISD/Results
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 768
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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