Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Anthony Kang
Date created: 2016-10-13 11:00:00
Date modified: 2016-10-14 09:55:32
phoA->LbCHI31->Flag tag
| Types | DnaRegion |
| Roles | engineered_region Translational_Unit |
| Sequences | BBa_K2090000_sequence (Version 1) |
Description
This linked protein encodes a Limonium bicolor-derived LbCHI31 chitinase enzyme with the signal sequence of phoA (alkaline phosphatase) linked upstream of the chitinase gene by a glycine-serine linker. Downstream from the LbCHI31 gene is a FLAG tag used for protein expression and production analysis via immunoprecipitation.The translated protein encodes a chitinolytic enzyme that can be secreted extracellularly by Escherichia coli; the presence of this protein can be identified using anti-FLAG antibodies.
Notes
Signal sequence peptides allow for secretion of linked proteins located downstream of the signal sequence; as such, the phoA signal had to be designed upstream of the LbCHI31. The FLAG tag must flank the 3' end of the genetic sequence for proper location on the translated protein for immunoprecipitation. The stop codons on phoA and LbCHI31 were eliminated, and a stop codon was added downstream of the FLAG tag. Similarly, ATG methionine was eliminated from LbCHI31 and only retained in phoA to initiate translation at the phoA site.Source
Alkaline phosphatase, or phoA, is a periplasmic protein in Escherichia coli that can be used by membrane vesicles in vitro for protein transportation.LbCHI31 originates from the Limonium bicolor genome; the coding sequence was taken from GenBank: ABD92819.1.