Difference between revisions of "Part:BBa K3617004"

Revision as of 22:33, 27 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 receptor-bound transmembrane domain (TMD). Thus, binding of the interleukin to the receptor results in a reconstitution of the two halves 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 transcription 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 cassette 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]

@@ Line 1: / Line 1: @@
-<!--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.
+<!--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 co-expressed 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.
-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.
+In parallel, we also express the Wsc1 TMD, which will be sorted and localized to the membrane. To this TMD, we will 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.
 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.
@@ Line 11: / Line 11: @@
 ==<partinfo>BBa_K3617004 short</partinfo>==
-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.
+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 receptor-bound transmembrane domain (TMD). Thus, binding of the interleukin to the receptor results in a reconstitution of the two halves 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 transcription factor LexA-VP16.
 ==<span class='h3bb'>Sequence and Features</span>==
 <partinfo>BBa_K3617004 SequenceAndFeatures</partinfo>
-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.
+This biobrick consists of multiple parts; An endoplasmic reticulum import signal peptide from the <i> Saccharomyces cerevisiae </i> cell wall integrity and stress response component 1 (Wsc1) receptor in <i> S. cerevisiae </i>, 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.
+The sequence was codon optimize for <i> S. cerevisiae </i>. The recognition sequences for SpeI, XbaI, NotI, EcoRI, PstI were avoided to follow the RFC10 standard.
 ==Structure and function==
-[[Image:T--UCopenhagen--il6-tev.png|500px|thumb|<p align="justify"> '''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.'''</p>]]
+[[Image:T--UCopenhagen--il6-tev.png|500px|thumb|<p align="justify"> '''figure 2: When the receptors trimerize with IL-6 extracellularly, the TEV protease is complemented and the transcription factor cassette LexA-VP16 is released by cleavage of the recognition sequence in the flexible linker, which triggers expression of the reporter gene.'''</p>]]
 ==Confocal flourescence microscopy==