Coding

Part:BBa_K1399007

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

GFP (mut3b) with SsrA-DAS+2 degradation tag


GFP (mut3b) (Part:BBa_E0040) with added engineered NYADAS-ssrA degradation tag (Part:BBa_M0051). 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 NYADAS tag encodes peptide sequence AANDENYNYDAS is reported to have low affinity to ClpX thus its mediated degradation very much depends on the concentration of SspB (ClpX mediator).[1] The two additional residues ‘NY’ extends tag between SspB and ClpX binding sites, thus preventing clash when both proteins are bound to the tag.[3] 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 [4].

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] McGinness, K. E., Baker, T. a & Sauer, R. T. Engineering controllable protein degradation. Mol. Cell 22, 701–7 (2006). [4] 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+2 degradation tag