BBa_K1399003

BBa_K1399003 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_K1399003
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Anna Stikane
Date created: 2014-09-18 11:00:00
Date modified: 2015-05-08 01:10:16

RFP from Discosoma striata (coral) with DAS-ssrA degradation tag



Types
DnaRegion

Roles
CDS

Coding

Sequences BBa_K1399003_sequence (Version 1)

Description

Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added DAS-ssrA degradation tag (part BBa_M0052). 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 AANDENYADAS 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 DAS tag is reported to have low affinity to ClpX thus its mediated degradation very much depends on the concentration of SspB (ClpX mediator).[1] However, be aware that exact protein degradation rate is influenced by multiple other factors: ClpXP and ClpAP protease 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 RFP 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 (AAV 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

RFP comes from part BBa_E1010, tag sequence was obtained from part BBa_M0052, but same tag was also used in paper by Andersen et al., (1998).[2]

Sequence Annotation Location Component / Role(s)
start
RFP
DAS-ssrA tag
stop
stop
1,3
4,675
676,708
709,711
712,714
start_codon feature/start
CDS feature/cds
tag feature/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_K1399003/1