Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Anna Stikane
Date created: 2014-09-17 11:00:00
Date modified: 2015-05-08 01:10:15
RFP from Discosoma striata (coral) with LVA-ssrA degradation tag
| Types | DnaRegion |
| Roles | Coding CDS |
| Sequences | BBa_K1399001_sequence (Version 1) |
Description
Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LVA-ssrA degradation tag. The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed.This tag is commonly attached to repressor proteins for use in various gene networks (e.g., oscillators).
The tag encodes peptide sequence AANDENYALVA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1]
The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LVA tag is reported to lead to fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; protein stability; Km of binding to the protease; temperature [3].
References:
[1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001).
[2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998).
[3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).