Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Justine Ring, Morgan Litschko, Malak Elbatarny, Joanna Semrau,
Date created: 2015-09-10 11:00:00
Date modified: 2016-01-27 10:39:40
Type III AFP with E-coil
| Types | DnaRegion |
| Roles | Coding CDS |
| Sequences | BBa_K1831003_sequence (Version 1) |
Description
What: This BioBrick is a fusion protein consisting of the E-coil domain of our coiled-coil system fused in-frame to the C-terminus of a Type III AFP. Type III AFP (PDB fIle: 1AME) is a moderately active antifreeze protein from the ocean pout, with a small (7Kb), globular structure. The E-coil refers to the coiled-coil with glutamic acid residues in the ???e??? and ???g??? positions of the helical wheel first used by Team Calgary 2013 (BBa_K1189011). This protein has been shown to be purified effectively by ice-affinity. If a His-tag is required for your purposes, see (BBa_K1831001).Why: The fusion of the E-coil to the Type III AFP will enable non-covalent, yet high affinity, specific attachment of AFPs to our protein scaffold and ???K??? coil BioBrick (BBa_K1831002). The interaction between the ???E??? and ???K??? coils involved oppositely charged residues, promoting the heterodimeric interaction of the coils that links AFPs to our scaffold. There is no His-tag, but ice-affinity purification can be used. By including a His-tag on our K-coil fusion protein (our scaffold: BBa_K1831002), and not the AFP with E-coil, we have enabled simple separation of any non-interacting coiled-coil domains.
Notes
The TypeIII AFP was fused in-frame to the E-coil through a gly-ser linker. The gly-ser coding DNA used the specific codons for a BamHI site, which is a unique cut site not found in either the AFP, E-coil, or the pSB1C3 backbone. Thus if you require the part in a different backbone, simple confirmation on cloning by a single restriction digest is possible.Source
The TypeIII AFP (PDB: 1AME) was obtained from Dr. Peter Davies lab at Queen's University. The E-coil was a novel sequence based off the E-coil (BBa_K1189012) and K-coil (BBa_K1189013) pair designed by Team Calgary 2013| Sequence Annotation | Location | Component / Role(s) |
| T7 rbs NdeI site Type III AFP E-coil BamHI site stop x2 XhoI site | 1,61 62,67 68,267 268,330 331,336 337,342 343,348 | promoter feature/promoter sequence_feature feature/misc feature/protein CDS feature/protein CDS feature/misc sequence_feature feature/stop stop_codon sequence_feature feature/misc |