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
VioB-AviTag [AviTagged violacein enzyme B]
| Types | DnaRegion |
| Roles | Coding CDS |
| Sequences | BBa_K597103_sequence (Version 1) |
Description
Avi-VioB codes for VioB with an appended biotinylation marker. When attached to a promoter, it can be biotinylated by E coii. 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 the violacein biosynthetic pathway with the added functionality of biotin-streptavidin binding.The VioB-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 activity of VioB.
The violacein biochemical pathway is composed of 5 enzmes, VioA-E. The violacein pathway involves five enzymes, VioA, VioB, VioC, VioD, and VioE, in the conversion of L-Tryptophan into a violacein, purple chromophore. However, only three of the five enzymes (VioA, VioB, and VioE) are required to produce a colored produce. Even with the exception of Vios C and D, L-Tryptophan is converted into prodeoxyviolacein, a green pigment.
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 from the parts registry.Source
VioB Part:BBa_K274004 (Cambridge 2009, Distribution 2011)Ref: Balibar CJ, Walsh CT (2006). "In Vitro Biosynthesis of Violacein from l-Tryptophan by the Enzymes VioA−E from Chromobacterium violaceum."
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Biochemistry Volume 45 Issue 51, Pages 15444-15457
Avitag: IDT DNA synthesized oligo single-stranded DNA annealed to form short double-stranded sequence.
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.