Difference between revisions of "Part:BBa K3617001"
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<partinfo>BBa_K3617001 short</partinfo> | <partinfo>BBa_K3617001 short</partinfo> | ||
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+ | This biobrick is a part of a two-protein system that is designed for detection of human IL-6 and transduction of the signal by means of a reconstituted ubiquitin. Development of split-ubiquitin as a tool for study of protein-protein interactions in vivo was first published in 1994 and has been an essential feature in biologists’ toolbox ever since (source: https://www.pnas.org/content/pnas/91/22/10340.full.pdf). A specific mutation in the N-terminal part protects it from binding spontaneously to the C-terminal part, however, reassociation can be facilitated by binding of a pair of proteins to which ubiquitin parts are fused. Human signal transducer gp130 was expressed in yeast for the first time in 1997 and further improvements paved the way to our own chimeric transmembrane proteins (source: https://pubmed.ncbi.nlm.nih.gov/9271090/). | ||
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+ | <h2><span class='h3bb'>Sequence and Features</span></h2> | ||
+ | <partinfo>BBa_K3617000 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 first and third domain of human soluble interleukin-6 co-receptor soluble glycoprotein 130 (sgp130), the transmembrane receptor of Wsc1 and the C-terminal part of the split version of ubiquitin, constituting aa. 363-405. Between the three domains of sgp130 and the transmembrane domain, a GGGGS-linker was added. Between the C-terminal split ubiquitin domain, two basic amino acids (KR), and the GGGGS-linker was added. | ||
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+ | A part of the protein constitutes of the C-terminal part (aa. 35-76) of the split ubiquitin molecule, which is fused to the reporter protein cassette LexA-VP16. Ubiquitin can be split into two parts, being the N-terminal and C-terminal ubiquitin that are able to reassociate in vivo to form active ubiquitin. This complementation was utilized so that upon interleukin-6 forming a heterotrimer with the gp130 domains of this biobrick and the IL-6 receptor domains of BBa_K3617000, the whole ubiquitin-molecule is formed, leading to activation of an intracellular deubiquitinase, thereby leading to release of Cub-bound LexA/VP16 . LexA is a DNA binding domain from Escherichia Coli (EG10533 (EcoCyc); P0A7C2 (UniProt)) and Herpes simplex virus Type 1 VP16 gene is a transcriptional activation domain. Upon complementation of split ubiquitin, the LexA-VP16 transcription factor is released and transported into the nucleus where it triggers reporter expression by promoter binding. | ||
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<p>This BioBrick is an ORF encoding a fusion protein consisting of:</p> | <p>This BioBrick is an ORF encoding a fusion protein consisting of:</p> | ||
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==Confocal flourescence microscopy== | ==Confocal flourescence microscopy== |
Revision as of 18:09, 24 October 2020
sgp130(D1-D3)-Cub
This biobrick is a part of a two-protein system that is designed for detection of human IL-6 and transduction of the signal by means of a reconstituted ubiquitin. Development of split-ubiquitin as a tool for study of protein-protein interactions in vivo was first published in 1994 and has been an essential feature in biologists’ toolbox ever since (source: https://www.pnas.org/content/pnas/91/22/10340.full.pdf). A specific mutation in the N-terminal part protects it from binding spontaneously to the C-terminal part, however, reassociation can be facilitated by binding of a pair of proteins to which ubiquitin parts are fused. Human signal transducer gp130 was expressed in yeast for the first time in 1997 and further improvements paved the way to our own chimeric transmembrane proteins (source: https://pubmed.ncbi.nlm.nih.gov/9271090/).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 130
Illegal BglII site found at 502
Illegal XhoI site found at 456 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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 first and third domain of human soluble interleukin-6 co-receptor soluble glycoprotein 130 (sgp130), the transmembrane receptor of Wsc1 and the C-terminal part of the split version of ubiquitin, constituting aa. 363-405. Between the three domains of sgp130 and the transmembrane domain, a GGGGS-linker was added. Between the C-terminal split ubiquitin domain, two basic amino acids (KR), and the GGGGS-linker was added.
A part of the protein constitutes of the C-terminal part (aa. 35-76) of the split ubiquitin molecule, which is fused to the reporter protein cassette LexA-VP16. Ubiquitin can be split into two parts, being the N-terminal and C-terminal ubiquitin that are able to reassociate in vivo to form active ubiquitin. This complementation was utilized so that upon interleukin-6 forming a heterotrimer with the gp130 domains of this biobrick and the IL-6 receptor domains of BBa_K3617000, the whole ubiquitin-molecule is formed, leading to activation of an intracellular deubiquitinase, thereby leading to release of Cub-bound LexA/VP16 . LexA is a DNA binding domain from Escherichia Coli (EG10533 (EcoCyc); P0A7C2 (UniProt)) and Herpes simplex virus Type 1 VP16 gene is a transcriptional activation domain. Upon complementation of split ubiquitin, the LexA-VP16 transcription factor is released and transported into the nucleus where it triggers reporter expression by promoter binding.
This BioBrick is an ORF encoding a fusion protein consisting of:
- The first 21 amino acids (Signal peptide for import to endoplasmatic reticulum) of the endogenous Cell wall integrity and stress response component 1 (Wsc1 [Jon: the protein is usually referred to as Wsc1 while the gene is called SLG1 what do we call it?) receptor in S. Cerevisiae.
- The first and third domain (D1-D3; aa 22-323) of human IL-6 co-receptor soluble glycoprotein 130 (sgp130).
- The transmembrane domain of Wsc1 (aa 327-351)
- C-terminal part of a split version of ubiquitin. (aa 363-405)
- Between the three domains of sgp130 and the transmembrane domain we added a GGGGS-linker and between the transmembrane domain (Wsc1) and the C-terminal split ubiquitin domain we added two basic amino acids; KR, and the GGGGS-linker again. [Jon: do you think we should explain the rational of these gene engineering choices or is that redundant?]
The C-terminal part (Cub) is the amino acids 35-76 of the split ubiquitin molecule and has the reporter protein cassette LexA-VP16 fused to Cub. LexA is a DNA binding domain from E. Coli (EG10533 (EcoCyc); P0A7C2 (UniProt)) and Herpes simplex virus Type 1 VP16 gene is a transcriptional activation domain.
The LexA-VP16 protein cassette is used in yeast two-hybrid method (Y2H) to assay protein-protein interaction (PPI), where the LexA-VP16 is dissociated from the Cub by deubiquitinase when PPI occurs. The LexA-VP16 transcription factor will then be transported into the nucleus where it will trigger the expression of reporter genes.
The sequence was codon optimized for S. cerevisiae using Benchling.[1]
Expected function of the protein
The signal peptide and transmembrane domain constitute the backbone of our modular framework for localizing our receptors at the plasma membrane as type I single pass transmembrane proteins. As a type I transmembrane protein the soluble interleukin receptor domains would be localized extracellularly while the N-terminal part of the split protein would be the intracellular. Ivanusic et al. (citation) introduced the use of the signal peptide and transmembrane domain in a split-ubiquitin system for screening for PPIs at the plasma membrane in S. cerevisiae. The two fibronectin type III soluble interleukin-6 receptor subunit alpha domains are mediating the binding of the receptor to interleukin-6 as seen in crystal structures (see fig. 1). The outer Ig-like domain of the receptor mediates other functions of the receptor (Vollmer et al. PMID: 10406952) and it is OMITTED in this part - this might cause the unwanted localization as addressed later! This BioBrick is intended to work together with (BioBrick) which has the outer three domains of the IL-6 co-receptor soluble glycoprotein 130 (sgp130), extracellularly and the C-terminal part of split ubiquitin intracellularly with the synthetic transcription factor linked to the C-terminal of the split ubiquitin domain. We hypothesized that BBa_K3617001 (this BioBrick) and BBa_K3617000 would both localize to the same membrane but that they would be dissociated in the absence of interleukin 6. In the presence of interleukin 6, we imagined that the extracellular domains of the two parts; IL-6R and sgp130, would associate into a heterotrimer consisting of IL-6, IL-6R and sgp130. Unfortunately, our assays indicated that the BioBricks do not work together as intended.
Sequence optimization
The sequence was codon optimized for S. cerevisiae, subsequently the sequence was modified by interchanging synonymous codons in the signal peptide region and in the flexible linkers and transmembrane domain to make the part fit into our modular framework where we can easily interchange intra- and extracellular domains while avoiding too long identical sequences which might cause unwanted homologous recombination. Furthermore we avoided following recognition sequences SpeI, XbaI, NotI, EcoRI, PstI to both follow the RFC10 standard and make the sequence useful for both USER cloning.
Confocal flourescence microscopy
Superfolding green flourescent protein was linked C-terminally to the protein and the cells where observed with confocal flourescence microscopy.
Most of the cells had either multiple or a single big flourescing aggregation positioned in the middle between the nucleus and the plasma membrane. This is most probably inclusion bodies. Especially for the cells that only had a single accumulation near the nucleus, we speculate that the protein might be stuck in the golgi apperatus. This would fit well with the findings of Vollmer et al. (DOI: 10.1046/j.1432-1327.1999.00511.x), that removing the N-terminal Ig-like domain of the IL-6R leads to retention in the secretory pathway and possible misfolding when expressing the IL-6R in P. pastoris. Next step in order to improve localization would be to put back the N-terminal Ig-domain.
Biosensor assays
To test the functionality of this part (BBa_K3617001), it was stably transformed into chromosome X (10) site 3 of S. cerevisiae and constitutively expressed by the pPCCW12 promoter together with BBa_K3617000 under constitutive expression by pTDH3 promoter and with nanoluciferase (citation??) under control by LexAop promoter. The cells were incubated at 30°C at a OD600=0,5 with different concentrations of commercial heterologously expressed IL-6 for 1, 3, 14 and 22 hours. We then did a luciferase assay by measuring luminescence after adding YeastBuster with 1% nanobit substrate to the samples.
We did not observe any correlation between interleukin-6 concentration and luminescence intensity at any incubation time. Instead, the amount of luminescence were in all cases very high compared to other strains that we measured - also for the mock experiment without any added interleukin-6. This might suggest that the two extracellular domains actually have an affinity for eachother even without the presence of IL-6, this would further imply that the two proteins, BBa_K3617001 and BBa_K3617000 are localizedd to the same subcellular compartments. Another possible explanation is that BBa_K3617001 is partially degraded after which the synthetic transcription factor is released and relocates to the nucleus. The degradation of BBa_K3617001 might even happen without any interaction with BBa_K361700. This could then be further investigated by integrating only the BBa_K3617001 and the reporter into the yeast and then doing another luciferase assay otherwise one could make a westernblot with anti-GFP on the strain used for the localization assays.
References
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