Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Malak Elbatarny, Joanna Semrau, Justine Ring, Morgan Litschko
Date created: 2015-08-31 11:00:00
Date modified: 2016-01-27 10:38:57
TypeIII and AFP E-coil fusion + His tag
| Types | DnaRegion |
| Roles | CDS Coding |
| Sequences | BBa_K1831001_sequence (Version 1) |
Description
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). There is a His-tag at the C-terminus of the ???E??? coil which provides an alternative purification method to ice-affinity.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. The addition of a His-tag to the C-terminus of the coil will allow for simple purification of the protein even for teams without access to an ice-affinity purification apparatus.
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 TypeIII AFP E-coil BamHI site His-tag XhoI site stop x2 | 1,61 62,67 68,267 268,330 331,336 337,351 358,363 352,357 | feature/promoter promoter feature/misc sequence_feature feature/protein CDS feature/protein CDS feature/misc sequence_feature feature/tag tag sequence_feature feature/misc stop_codon feature/stop |