Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: James Mathew
Date created: 2011-09-25 11:00:00
Date modified: 2015-05-08 01:12:50
GFP-AviTag [AviTagged green fluorescent protein]
| Types | DnaRegion |
| Roles | Coding CDS |
| Sequences | BBa_K597101_sequence (Version 1) |
Description
GFP-avitag codes for green fluorescent protein with an appended biotinylation marker. When attached to a promoter, it can be used as a fluorescent reporter and biotinylated by E coli. E coli has a natural mechanism encoded by the BirA gene to produce biotin and biotinylate proteins - this mechanism can be enhanced by culturing with biotin. This enzyme can then be used in biotin-streptavidin binding applications.The GFP-avitag fusion protein was constructed to have the AviTag sequence on the C-terminus, followed by a stop codon. The resulting protein, once biotinylated, is capable of a ligand reaction with surface-coated avidin, while retaining the fluorescence of GFP. This molecule was used to test the efficacy of the binding interaction between the functionalized protein and a streptavidin-coated surface.
Notes
The avitag is capable of being on either the C or N terminus of the protein of interest with little difference between the two in the binding efficiency or expression of the protein. We chose to put the Avitag on the C-terminus for the convenience of using a primer dimer pair to insert the iGEM suffix to the sample provided by the Delisa Lab Group.Source
GFP aliquot was provided by Dr. DeLisa (Cornell University, Chemical Engineering Department) in the form of a pZE12-SMCS vector.AviTag: Avidity, L.L.C., Eleanor Roosevelt Institute, Denver, Colorado 80206, USA.
Ref: Cull MG, Schatz PJ (2000). ???Biotinylation of proteins in vivo and in vitro using small peptide tags.???
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Methods of Enzymology Vol 326, 2000, Pages 430-440.