Coding

Part:BBa_K1399004

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

GFP (mut3b) with LVA-ssrA degradation tag


GFP (mut3b) ( 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 (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 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).


Contribution

Group: Valencia_UPV iGEM 2018

Authors: Adrián Requena, Carolina Ropero

Part BBa_K2656013 is the GFPmut3b coding sequence with the LVA degradation tag. It is the BBa_K1399004 original sequence adapted to be Golden Gate compatible. Thus, it can be used with Biobrick and Golden Braid assembly methods.


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
//cds/reporter/gfp
//chassis/prokaryote/bsubtilis
//chassis/prokaryote/ecoli
//function/reporter/fluorescence
Parameters
colorGreen
directionForward
emission
emit511
excitation
excite501
kegg
lum
proteinGFPmut3b
swisspro
tagSsrA-LVA degradation tag