Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Anna Stikane
Date created: 2014-09-16 11:00:00
Date modified: 2015-05-08 01:10:15
RFP from Discosoma striata (coral) with AAV-ssrA degradation tag
| Types | DnaRegion |
| Roles | Coding CDS |
| Sequences | BBa_K1399000_sequence (Version 1) |
Description
Mutant RFP from Discosoma striata (coral) (see part E1010) with added AAV-ssrA degradation tag (part I11012). 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.The tag encodes peptide sequence AANDENYAAAV 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 AAV tag is reported to lead to reasonably fast protein degradation, degrading GFP with rate -0.012 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: 1) ClpXP and ClpAP protease and SspB mediator concentrations; 2) Protein stability; 3) Km of binding to the protease; 4) Temperature [3].
[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).