Coding

Part:BBa_K1399002

Designed by: Anna Stikane   Group: iGEM14_Edinburgh   (2014-09-19)

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


Mutant RFP from Discosoma striata (coral) (Part:BBa_E1010) with added LAA-ssrA degradation tag (Part:BBa_M0050). 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 AANDENYALAA 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 (Figure 1).[1]

Figure 1 SsrA degron mediated protein degradation. (A) Any version of SsrA tags can be attached to any protein of interest (e.g. RFP or GFP). (B) The tag is recognized by ClpX unfoldase forming a complex with ClpP protease and the tagged protein gets degraded.

The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is E. coli wild type ssrA tag and is reported to lead to very 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).


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 555
    Illegal AgeI site found at 667
  • 1000
    COMPATIBLE WITH RFC[1000]
[edit]
Categories
//cds/reporter/chromoprotein
//chassis/prokaryote/bsubtilis
//chassis/prokaryote/ecoli
//function/reporter
//function/reporter/fluorescence
Parameters
abs
biology
colorRed
directionForward
emission607
emit607
excitation584
excite584
kegg
lum
proteinmRFP1
swisspro
tagSsrA-LAA degradation tag