Coding

Part:BBa_K3617004

Designed by: Emil Funk Vangsgaard   Group: iGEM20_UCopenhagen   (2020-10-23)
Revision as of 23:02, 25 October 2020 by EmilFunk (Talk | contribs)


sIL-6R-nTEV

This biobrick is a part of a 2-protein system that is designed for detection of human interleukin-6 and transduction of the signal by means of a reconstitution of a split TEV protease. The split TEV was originally developed by Wehr, M. C. et al. In 2006[[#References[1]]] for measuring protein-protein interaction. This part resembles that of sIL-6R-Nub in which the extrecellular receptor remains the human interleukin-6 receptor, but now with a split TEV protease, an N-terminal part (amino acid residues 1-118) and a C-terminal part (amino acid residues 119-242), fused with a flexible linker on the C-terminal of the receptorbound transmembrane domain (TMD). Thus, binding of the interleukin to the receptor results in a reconstitution of the two halfes nTEV and cTEV, thus rendering it active. The activated protease will cleave a recognition site on a flexible linker bound to another TMD, which releases the synthetic transciption factor LexA-VP16.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 130
    Illegal BglII site found at 502
    Illegal XhoI site found at 456
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

This biobrick consists of multiple parts; An endoplasmic reticulum import signal peptide from the Saccharomyces cerevisiae cell wall integrity and stress response component 1 (Wsc1) receptor in S. cerevisiae , the second and third domain of human soluble interleukin-6 receptor subunit alpha (sIL-6R), the transmembrane receptor of Wsc1 and the N-terminal part of the split TEV protease, constituting the amino acid residues 1-118. Between the sIL-6R domains and the transmembrane domain, a flexible 2XXGGGGS linker exists. Between the transmembrane domain and the N-terminal split ubiquitin domain two basic amino acids (KR) have been added together with the 2XGGGGS linker.

Sequence optimization

The sequence was codon optimize for S. cerevisiae. The recognition sequences for SpeI, XbaI, NotI, EcoRI, PstI were avoided to follow the RFC10 standard.

Structure and function

figure 2: When the receptors trimerize with IL-6 extracellularly, the TEV protease is complemented and the transcription factor casette LexA-VP16 is released by cleavage of the recognition sequence in the flexible linker, which triggers expression of the reporter gene.

Confocal flourescence microscopy

References

[1] Wehr, M. C., Laage, R., Bolz, U., Fischer, T. M., Grünewald, S., Scheek, S., Bach, A., Nave, K. A., & Rossner, M. J. (2006). Monitoring regulated protein-protein interactions using split TEV. Nature Methods, 3(12), 985–993. https://doi.org/10.1038/nmeth967

[2]

[3]


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