Coding

Part:BBa_K1399008

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

GFP (mut3b) with DAS-ssrA degradation tag


GFP (mut3b) (see Part:BBa_E0040) with added DAS-ssrA degradation tag (see Part:BBa_M0052). 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. SsrA tags encode peptide sequence that is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB (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.

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 encodes peptide sequence AANDENYADAS 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).

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 644
[edit]
Categories
//chassis/prokaryote/bsubtilis
//chassis/prokaryote/ecoli
//function/reporter
//function/reporter/fluorescence
Parameters
colorGreen
directionForward
emission
emit511
excitation
excite501
kegg
lum
proteinGFPmut3b
swisspro
tagSsrA-DAS degradation tag