Difference between revisions of "Part:BBa K3617004"
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<!--Our intermediate design utilizes the same receptor-system as the ubiquitin-based design, but with few modifications. Thanks to our supervisor’s guidance, we were introduced to another kind of split-protein: the split TEV-protease. This was another method developed by Wehr, M. C. et al. In 2006 to monitor protein-protein interactions. Here, we again have two engineered inactive halves of the TEV-protease, that only regain activity when coexpressed as fusion constructs with interacting proteins . Therefore, we again utilize the receptor/TMD domains from the previous designs, but now each of our receptors will be fused to one half of the TEV-protease instead with a flexible linker.
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In parallel, we also express the Wsc1 TMD, which will be sorted and localized to the membrane. To this TMD, we’ll fuse the same transcription factor from the previous design (LexA-VP16), and use the recognition sequence for the TEV-protease as the linker between the two. This means that the TEV-protease, upon reconstitution, will be able to cleave the transcription factor and free it into the cytosol. In theory, the TEV-protease will be able to cut many transcription factors loose, meaning that one interleukin (by extension of the association of our two receptors) will result in the cleavage of multiple transcription factors and thus an amplification of the signal.
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Again, this extra amplification step made us hopeful, but in order to achieve the highest level of amplification possible we moved on to other venues.
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Simultaneously, we started modeling our pathways in the dry lab.
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__NOTOC__
 
__NOTOC__
 
==<partinfo>BBa_K3617004 short</partinfo>==
 
==<partinfo>BBa_K3617004 short</partinfo>==
  
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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 [[Part:BBa_K3617000|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.
  
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<h2><span class='h3bb'>Sequence and Features</span></h2>
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==<span class='h3bb'>Sequence and Features</span>==
 
<partinfo>BBa_K3617004 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3617004 SequenceAndFeatures</partinfo>
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This biobrick consists of multiple parts; An endoplasmatic 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 version of ubiquitin, constituting the first 34 amino acids of ubiquitin. The domain possesses the Ile13Gly mutation which inhibits the spontaneous association of the two split protein halves by reducing their affinity to each other. Between the sIL-6R domains and the transmembrane domain, a flexible 2XXGGGGS linker [[#References|[3]]] 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.
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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 version of ubiquitin, constituting the first 34 amino acids of ubiquitin. The domain possesses the Ile13Gly mutation which inhibits the spontaneous association of the two split protein halves by reducing their affinity to each other. Between the sIL-6R domains and the transmembrane domain, a flexible 2XXGGGGS linker [3] 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==
 
==Sequence optimization==
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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==
 
==Structure and function==
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==Confocal flourescence microscopy==
 
==Confocal flourescence microscopy==
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===References===
 
===References===
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<p>[1] Johnsson, N., & Varshavsky, A. (1994). Split ubiquitin as a sensor of protein interactions in vivo. Proceedings of the National Academy of Sciences of the United States of America, 91(22), 10340–10344. https://doi.org/10.1073/pnas.91.22.10340 https://www.pnas.org/content/pnas/91/22/10340.full.pdf</p>
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<p> [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 </p>
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<p>[2] Vollmer, P., Oppmann, B., Voltz, N., Fischer, M., & Rose-John, S. (n.d.). 438±446 (1999) q FEBS 1999. In Eur. J. Biochem (Vol. 263). https://www.sciencedirect.com/science/article/abs/pii/S0022175996001639 </p>
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<p> [2]  </p>
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<p>[3] Chen, X., Zaro, J. L., & Shen, W. C. (2013). Fusion protein linkers: Property, design and functionality. In Advanced Drug Delivery Reviews (Vol. 65, Issue 10, pp. 1357–1369). https://doi.org/10.1016/j.addr.2012.09.039</p>
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<p> [3] </p>
  
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<!-- Uncomment this to enable Functional Parameter display  
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<!-- Uncomment this to enable Functional Parameter display
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K3617000 parameters</partinfo>
 
<partinfo>BBa_K3617000 parameters</partinfo>
 
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Revision as of 22:13, 25 October 2020


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 version of ubiquitin, constituting the first 34 amino acids of ubiquitin. The domain possesses the Ile13Gly mutation which inhibits the spontaneous association of the two split protein halves by reducing their affinity to each other. Between the sIL-6R domains and the transmembrane domain, a flexible 2XXGGGGS linker [3] 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

Confocal flourescence microscopy

Biosensor assays

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]