BBa_K2090001

BBa_K2090001 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_K2090001
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: TP_CC-SanDiego 2016
Date created: 2016-10-13 11:00:00
Date modified: 2016-10-14 09:44:18

phoA->LbCHI32->Flag tag



Types
DnaRegion

Roles
engineered_region

Translational_Unit

Sequences BBa_K2090001_sequence (Version 1)

Description

This linked protein encodes a Limonium bicolor-derived LbCHI32 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 LbCHI32. 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 LbCHI32 were eliminated, and a stop codon was added downstream of the FLAG tag. Similarly, ATG methionine was eliminated from LbCHI32 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: ACE79211.1.

Sequence Annotation Location Component / Role(s)
phoA
start
LbCHI32
stop
FLAG
1,63
1,3
78,1035
1066,1068
1041,1065
sequence_feature feature/misc
start_codon feature/start
CDS feature/protein
stop_codon feature/stop
tag feature/tag
igem#experience
None
 
igem#status
Planning
 
synbiohub#ownedBy
user/james
 
synbiohub#ownedBy
user/myers
 
synbiohub#topLevel
BBa_K2090001/1