BBa_K1399004

BBa_K1399004 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_K1399004
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Anna Stikane
Date created: 2014-09-18 11:00:00
Date modified: 2015-06-01 07:07:29

GFP (mut3b) with LVA-ssrA degradation tag



Types
DnaRegion

Roles
CDS

Coding

Sequences BBa_K1399004_sequence (Version 1)

Description

GFP (mut3b) (see part BBa_E0040) with added LVA-ssrA degradation tag. The tag increases GFP turn-over rate, thus providing better temporal resolution of green 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 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).

Notes

The tag was attached to GFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additonal residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (LVA in this case) are recognised by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.

Source

GFP comes from part E1010, the tag sequence was obtained from paper by Andersen et al., (1998).[2]

Sequence Annotation Location Component / Role(s)
start
GFP (mut3b)
LVA
stop
stop
1,3
4,714
715,747
748,750
751,753
start_codon feature/start
CDS feature/cds
feature/tag tag
feature/stop stop_codon
stop_codon feature/stop
igem#experience
Works
 
igem#sampleStatus
In stock
igem#status
Available
 
synbiohub#ownedBy
user/james
 
synbiohub#ownedBy
user/myers
 
synbiohub#topLevel
BBa_K1399004/1