Team:Stanford-Brown-RISD/Parts
Stanford-Brown-RISD Key Parts (32 submitted in total)
| BBa_K2868000 | Composite Part T7 CsgA-6xhistag expression |
| Designed by Cale Lester. This composite part consists of a T7 promoter with RBS (BBa_K525998), followed by a coding region for csgA (BBa_K1583000) with a His-Tag expressed at the end, an ochre stop codon, and then a BglII cut site followed by a T7 terminator (BBa_K731721). The construct constitutively produces the amyloid-forming E.coli curlin-precursor protein csgA when restriction ligated into a backbone plasmid of choice. The his-tag allows for the protein product to be purified with standard his-tag protein purification methods. Because of the T7 promoter and RBS, for protein production this construct must be used in T7 cell lines like BL21 that produce T7 RNA polymerase. The SalI and BglII cut sites also allow for the replacement with a desired coding region after the construct is successfully in a plasmid backbone. | |
| BBa_K2868011 | Composite Part T7 CBD-6xhistag expression |
| Designed by Cale Lester. This part contains the genetic machinery to produce a chitin binding protein that is linked with a his-tag, with a T7 promoter and TE1 terminator. | |
| BBa_K2868014 | HHTC-Re |
| Designed by Advait Patil. This is a version of the peptide computationally designed by Kozisek et al. (Chemistry, 2008) to bind to copper (HHTC). This metal binding domain has had amino acids replaced for specificity, and has a flexible GSGGSG linker attached to allow for spacing for proper folding of the constituent domains in any fusion proteins being created. | |
| BBa_K2868015 | Composite Part HHTC_RE-CBD x2 Copper Binding Expression |
| Designed by Advait Patil. This is a fusion protein formed with a Chitin Binding Domain (CBD) and semi-rationally designed copper binding domain (HHTC-Re) as subunits. There are two metal binding domains, allowing for two copper atoms to be bound to each protein molecule. GSGGSG linkers were added in between the CBD and HHTC-Re subunits to spatially isolate the individual domains while still allowing for flexibility. | |
| BBa_K2868018 | Composite Part HHTC_RE-CBD x3 Copper Binding Expression |
| Designed by Advait Patil. This is a fusion protein formed with a Chitin Binding Domain (CBD) and semi-rationally designed copper binding domain (HHTC-Re) as subunits. There are three metal binding domains, allowing for three copper atoms to be bound to each protein molecule. GSGGSG linkers were added in between the CBD and HHTC-Re subunits to spatially isolate the individual domains while still allowing for flexibility. | |
| BBa_K2868019 | Composite Part HHTC_RE-CBD x6 Copper Binding Expression |
| Designed by Advait Patil. This is a fusion protein formed with a Chitin Binding Domain (CBD) and semi-rationally designed copper binding domain (HHTC-Re) as subunits. There are six metal binding domains, allowing for six copper atoms to be bound to each protein molecule. GSGGSG linkers were added in between the CBD and HHTC-Re subunits to spatially isolate the individual domains while still allowing for flexibility. | |
| BBa_K2868021 | Composite Part FP151 6xhistag T7 expression |
| Designed by Cale Lester. An IPTG inducible T7 promoter, then medium strength RBS, then a SalI cut-site, for the mfp151 DNA sequence which has an added 6x polyhistidine tag at the end, followed by a taa stop codon, then a bglII cut-site, and finally a and a T7 terminator. | |
| BBa_K2868024 | Composite Part Csga-CBD-6xhistag Fusion T7 expression |
| Designed by Cale Lester. 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. | |
| BBa_K2868028 | Composite Part CBD4x T7 expression |
| Designed by Cale Lester. T7 promoter, a salI cut-site, then a start codon then a Flag, Lumio, 6x polyhisitine tag, followed by Bacillus circulans chitin binding domain, flexible linker, S. griseus 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. | |
| BBa_K2868032 | Composite Part Tyrosinase+Co-Factor Arabinose induced Co-expression |
| Designed by Cale Lester. Arabinose inducible promoter, medium RBS, his tagged tyrosinase coding, stop codon, tactag spacer, medium strength RBS, co-factor his-tagged coding region, stop codon, T7 terminator. |
Validated 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