| Types | DnaRegion
|
| Roles | engineered_region
Composite
|
| Sequences | BBa_K1251000_sequence (Version 1)
|
Description
This is a composite part consisting of a T7 promoter containing a lac operator, a RBS, the coding sequence for the antifreeze protein, RiAFP, a 6x His tag, a secretion peptide, and a double terminator. This part controls the expression of RiAFP. The expressed protein is tagged with an amino acid motif that consists of six histidine (His) residues fused to the C-terminus of RiAFP. Histidine tags are widely used because they are small and rarely interfere with the function, activity, or structure of target proteins. The polyhistidine-tag is to be used to detect the secreted protein via anti-polyhistidine-tag antibodies or alternatively by in-gel staining (SDS-PAGE) with fluorescent probes bearing metal ions. The HlyA is a signal peptide found in the C-terminal signal sequence of alpha-hemolysin (HlyA). It is used to target RiAFP for secretion via the Type I secretion pathway of gram-negative bacteria. Fusion of the HlyA signal peptide to RiAFP results in transport of the protein from the cytoplasm to the extracellular medium in a single step
Notes
The project undertaken by the Cambridge School of Weston (CSW) 2014 iGEM team builds upon the work of the 2009 Utah State team that developed a protein secretion mechanism and that of the 2011 Yale team which synthesized an ???antifreeze??? protein (RiAFP) isolated from a cold-tolerant beetle called Rhagium inquisitor. The
Source
The T7 promoter is a BioBrick part BBa_1712074. The HlyA signal peptide is BioBrick pat BBa_K208006. BBa_B0015 is a double terminator (B0010-B0012). The DNA sequence for RiAFP comes from GeneBank.
