Coding

Part:BBa_K1399003:Design

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


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


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]


Design 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 (DAS in this case) have low affinity to 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]

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).