Difference between revisions of "Part:BBa K3617000"
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<partinfo>BBa_K3617000 short</partinfo>
 
<partinfo>BBa_K3617000 short</partinfo>
  
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This biobrick is an ORF encoding a fusion protein consisting of :
 
−
<ul>
 
−
<li>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 refered to as Wsc1 while the gene is called SLG1 what do we call it?) receptor in S. Cerevisiae. </li>
 
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<li> The second and third domain (aa 115-365) of human soluble interleukin-6 receptor subunit alpha (sIL-6R).</li>
 
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<li> The transmembrane domain of Wsc1</li>
 
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<li> N-terminal part of a split version of ubiquitin. This means the first 34 amino acids of the ubiquitin protein. Compared to Wt ubiquitin, the domain has a mutation(Ile13Gly). This mutation inhibits the spontaneous association of the two split protein halves by reducing the affinity. </li>
 
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<li> Between the sIL-6R domains and the transmembrane domain we added a flexible 2XGGGGS linker (cite: PMID: 23026637) and between the transmembrane domain and the n-terminal split ubiquitin domain we added two basic amino acids; KR, and the 2XGGGGS linker again. </li>
 
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</ul>
 
  
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<h4>Expected function of the protein: </h4>
 
  
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This part is ment to function as a IL-6 receptor together with <bbpart>BBaK3617001</bbpart>. Compared to the human receptor the extracellular domains are severely truncated and the intracellular domains are replaced with our alternative signal transduction designs.
+
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 reconstituted ubiquitin. It is mainly comprised of the extracellular part of the human soluble interleukin-6 receptor and of the N-terminal part of split 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 the split-ubiquitin parts are fused. Human interleukin-6 receptor was expressed in yeast for the first time in 1996 and further improvements paved the way to our own chimeric transmembrane proteins (source: https://www.sciencedirect.com/science/article/abs/pii/S0022175996001639).
  
−
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 of the receptor (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 <bbpart>BBaK3617001</bbpart>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_K3617000 (this biobrick) and <bbpart>BBaK3617001</bbpart>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. The trimerization would cause intracellular complementation of the ubiquitin that can then be recognized by an endogenous deubiquitinizing enzyme which releases the transcription factor resulting in expression of a reporter. Unfortunately, our assays indicated that the biobricks do not work together as intended.
 
  
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<h4>Sequence optimization</h4>
+
<h2><span class='h3bb'>Sequence and Features</span></h2>
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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.
+
<partinfo>BBa_K3617000 SequenceAndFeatures</partinfo>
  
 +
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 (cite: PMID: 23026637) 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.
  
−
<!-- Add more about the biology of this part here
+
<h2>structure and function<h2>
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===Usage and Biology===
+
−
<!-- -->
+
  
−
<h2><span class='h3bb'>Sequence and Features</span></h2>
+
This part is designed to function as a human IL-6 receptor together with BBaK3617001. Compared to the endogenous? human receptor, the extracellular domains are severely truncated, and the intracellular domains are replaced with our alternative signal transduction designs (I don’t know what this is).
−
<partinfo>BBa_K3617000 SequenceAndFeatures</partinfo>
+
  
−
<h2><b>Confocal flourescence microscopy</b></h2>
+
The signal peptide and transmembrane domain constitute the backbone of the modular framework of the UCopenhagen 2020 team (CIDosis). These are used for localizing interleukin-1, interleukin-6 and interleukin-10 receptors at the plasma membrane of S. cerevisiae as type I single pass transmembrane proteins. As a type I transmembrane protein, the soluble interleukin receptor domains localizes extracellularly while the N-terminal part of the split protein is 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.
  
−
Superfolding green flourescent protein was linked C-terminally to the protein and the cells where observed with <span>confocal flourescence microscopy.</span>
+
The two fibronectin type III soluble interleukin-6 receptor subunit alpha domains mediates the binding of the receptor to interleukin-6, as demonstrated on figure 1. The outer Ig-like domain of the receptor mediates other functions of the receptor (Vollmer et al. (which year) PMID: 10406952).
 +
 
 +
This biobrick is intended to work together with BBaK3617001 and constitute a functional human IL-6 receptor. BBaK3617001 possesses outer three domains of the IL-6 co-receptor soluble glycoprotein 130 (sgp130), extracellularly and the C-terminal part of split ubiquitin intracellularly with a synthetic transcription factor linked to the C-terminal of the split ubiquitin domain. The synthetic transcription factor is a fusion of a DNA binding domain of the LexA transcription facter from Escherichia Coli and an activation domain from the herpes simplex virus transcriptional regulatory protein VP16. LexA-VP16 is often used in yeast 2 hybrid assays as it is not endogenous to S. cerevisiae, thus providing orthogonality. After translocation into the nucleus, LexA-VP16 binds to a synthetic promoter with repeats of the lexo (LexO?) region from E. coli. It is thought that the biobrick and BBaK3617001 localize to the same membrane and will be dissociated in the absence of interleukin-6. In the presence of interleukin-6, the extracellular domains of the two parts; IL-6R and sgp130 associate into a heterotrimer consisting of IL-6, IL-6R and sgp130. The trimerization then causes intracellular complementation of the ubiquitin that is recognized by an endogenous deubiquitinizing enzyme which releases the transcription factor, resulting in expression of a reporter gene.
 +
 
 +
 
 +
<h2>Sequence optimization<h2>
 +
The sequence was codon optimized for S. cerevisiae. The recognition sequences for SpeI, XbaI, NotI, EcoRI, PstI were avoided to follow the RFC10 standard.
 +
 
 +
 
 +
<h2><b>Confocal flourescence microscopy</b></h2>
 +
In order to investigate the localization of our protein, superfolding green flourescent protein was linked to the C-terminal of the protein product of the biobrick. Consequently, the cells were observed with confocal flourescence microscopy for visualization.
  
 
[[Image:T--UCopenhagen--results-localization22 1flour.jpg|700px|thumb|center|<p align="justify"> '''figure 3a: Pictures were taken with a 150 &mu;m pinhole. The image shows both a faint localisation in endoplasmatic reticulum, and at the membrane, but most of the protein ends up in inclusion bodies/vacoules'''</p>]]
 
[[Image:T--UCopenhagen--results-localization22 1flour.jpg|700px|thumb|center|<p align="justify"> '''figure 3a: Pictures were taken with a 150 &mu;m pinhole. The image shows both a faint localisation in endoplasmatic reticulum, and at the membrane, but most of the protein ends up in inclusion bodies/vacoules'''</p>]]
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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.
+
The majority of investigated cells had either multiple or a single big fluorescent aggregate. This aggregate was positioned between the nucleus and the plasma membrane and can likely be attributed to the presence of inclusion bodies. It is possible that the protein may be stuck in the golgi apparatus, which is especially evident for the cells that had only one accumulation near their nucleus, in accordance to previous findings of Vollmer et al. (year) that have shown that removing the N-terminal Ig-like domain of the IL-6 receptor leads to retention of the protein in the secretory pathway. To circumvent this localization issue, one could add back the N-terminal Ig-domain of the IL6-R.
  
 
<h2>Biosensor assays</h2>
 
<h2>Biosensor assays</h2>
  
−
To test the functionality of this part (BBa_K3617000), it was stably transformed into chromosome x site 3 of S. cerevisiae and constitutively expressed by the pTDH3 promoter together with <bbpart>BBa_K3617001</bbpart> under constitutive expression by pPCCW12 promoter and with nanoluciferase (citation??) under control by lexAop promoter. The cells were incubated at 30&deg;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.  
+
To test the functionality of the part, it was stably transformed into chromosome x site 3 of S. cerevisiae and constitutively expressed by the pTDH3 promoter together with BBa_K3617001. The latter was under constitutive expression by the pPCCW12 promoter. Together, they were also expressed with a NanoBit luciferase, which luminesces 100 times brighter than firefly or Renilla luciferase. The luciferase expression was controlled by binding of lexA-VP16 to the lexo 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. A luminescence assay was performed to analyze the expression of luciferase after application of an industrial extraction reagent called YeastBuster to the samples, which allows for fast extraction of native proteins from yeast without mechanical disruption and enzymatic lysis, mixed with NanoBiT substrate.  
 +
 
  
 
[[Image:T--UCopenhagen--results-IL6assay.jpg|700px|thumb|center|<p align="justify"> '''figure 4: Luciferase assay. '''</p>]]
 
[[Image:T--UCopenhagen--results-IL6assay.jpg|700px|thumb|center|<p align="justify"> '''figure 4: Luciferase assay. '''</p>]]
  
−
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_K3617000 and <bbpart>BBa_K3617001</bbpart> are localizedd to the same subcellular compartments. Another possible explanation is that <bbpart>BBa_K3617001</bbpart> is partially degraded after which the synthetic transcription factor is released and relocates to the nucleus. The degradation of <bbpart>BBa_K3617001</bbpart> might even happen without any interaction with BBa_K361700. This could then be further investigated by integrating only the <bbpart>BBa_K3617001</bbpart> 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.
+
No correlation between IL-6 concentration and luminescence intensity was observed at any incubation time. This indicates, that the biosensor does not work as intended for the concentrations and experimental conditions of the experiment. A similar assay was performed with the IL-10 biosensor strain also developed by the UCopenhagen 2020 team (link to biobricks). Compared with the IL-10 biosensor, the amount of luminescence was between 3-10 times higher at all concentrations and incubation times.
 +
 
 +
[[Image:T--UCopenhagen--results-IL10assay.jpg|700px|thumb|center|<p align="justify"> '''figure 4: Luciferase assay. '''</p>]]
 +
 
 +
This suggests that the two extracellular domains have an affinity to each other even without the presence of IL-6. This further implies that the proteins produced from BBa_K3617000 and BBa_K3617001 are localized at the same subcellular compartment(s). The high amount of luminescence may also be caused by partial degradation of BBa_K3617001 may also be partially degraded, after which the synthetic transcription factor, lexA-VP16, is released and re-localizes to the nucleus. This could be verified by integrating only the BBa_K3617001 and reporter gene into S. cerevisiae and performing an additional luciferase assay. Alternatively, one may perform a western blot with primary antibody against GFP on the strain used for the localization assays.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 17:32, 24 October 2020


sIL-6R-Nub


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 reconstituted ubiquitin. It is mainly comprised of the extracellular part of the human soluble interleukin-6 receptor and of the N-terminal part of split 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 the split-ubiquitin parts are fused. Human interleukin-6 receptor was expressed in yeast for the first time in 1996 and further improvements paved the way to our own chimeric transmembrane proteins (source: https://www.sciencedirect.com/science/article/abs/pii/S0022175996001639).


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 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 (cite: PMID: 23026637) 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.

structure and function<h2> This part is designed to function as a human IL-6 receptor together with BBaK3617001. Compared to the endogenous? human receptor, the extracellular domains are severely truncated, and the intracellular domains are replaced with our alternative signal transduction designs (I don’t know what this is). The signal peptide and transmembrane domain constitute the backbone of the modular framework of the UCopenhagen 2020 team (CIDosis). These are used for localizing interleukin-1, interleukin-6 and interleukin-10 receptors at the plasma membrane of S. cerevisiae as type I single pass transmembrane proteins. As a type I transmembrane protein, the soluble interleukin receptor domains localizes extracellularly while the N-terminal part of the split protein is 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 mediates the binding of the receptor to interleukin-6, as demonstrated on figure 1. The outer Ig-like domain of the receptor mediates other functions of the receptor (Vollmer et al. (which year) PMID: 10406952). This biobrick is intended to work together with BBaK3617001 and constitute a functional human IL-6 receptor. BBaK3617001 possesses outer three domains of the IL-6 co-receptor soluble glycoprotein 130 (sgp130), extracellularly and the C-terminal part of split ubiquitin intracellularly with a synthetic transcription factor linked to the C-terminal of the split ubiquitin domain. The synthetic transcription factor is a fusion of a DNA binding domain of the LexA transcription facter from Escherichia Coli and an activation domain from the herpes simplex virus transcriptional regulatory protein VP16. LexA-VP16 is often used in yeast 2 hybrid assays as it is not endogenous to S. cerevisiae, thus providing orthogonality. After translocation into the nucleus, LexA-VP16 binds to a synthetic promoter with repeats of the lexo (LexO?) region from E. coli. It is thought that the biobrick and BBaK3617001 localize to the same membrane and will be dissociated in the absence of interleukin-6. In the presence of interleukin-6, the extracellular domains of the two parts; IL-6R and sgp130 associate into a heterotrimer consisting of IL-6, IL-6R and sgp130. The trimerization then causes intracellular complementation of the ubiquitin that is recognized by an endogenous deubiquitinizing enzyme which releases the transcription factor, resulting in expression of a reporter gene. <h2>Sequence optimization<h2> The sequence was codon optimized for S. cerevisiae. The recognition sequences for SpeI, XbaI, NotI, EcoRI, PstI were avoided to follow the RFC10 standard. <h2>Confocal flourescence microscopy

In order to investigate the localization of our protein, superfolding green flourescent protein was linked to the C-terminal of the protein product of the biobrick. Consequently, the cells were observed with confocal flourescence microscopy for visualization.

figure 3a: Pictures were taken with a 150 μm pinhole. The image shows both a faint localisation in endoplasmatic reticulum, and at the membrane, but most of the protein ends up in inclusion bodies/vacoules

figure 3b: Pictures were taken with a 150 μm pinhole. Here the inclusion bodies are also evident in the brightfield image.

figure 3c: Pictures were taken with a 150 μm pinhole. Flourescence in inclusion bodies and very faintly at membrane and around nucleus


The majority of investigated cells had either multiple or a single big fluorescent aggregate. This aggregate was positioned between the nucleus and the plasma membrane and can likely be attributed to the presence of inclusion bodies. It is possible that the protein may be stuck in the golgi apparatus, which is especially evident for the cells that had only one accumulation near their nucleus, in accordance to previous findings of Vollmer et al. (year) that have shown that removing the N-terminal Ig-like domain of the IL-6 receptor leads to retention of the protein in the secretory pathway. To circumvent this localization issue, one could add back the N-terminal Ig-domain of the IL6-R.

Biosensor assays

To test the functionality of the part, it was stably transformed into chromosome x site 3 of S. cerevisiae and constitutively expressed by the pTDH3 promoter together with BBa_K3617001. The latter was under constitutive expression by the pPCCW12 promoter. Together, they were also expressed with a NanoBit luciferase, which luminesces 100 times brighter than firefly or Renilla luciferase. The luciferase expression was controlled by binding of lexA-VP16 to the lexo 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. A luminescence assay was performed to analyze the expression of luciferase after application of an industrial extraction reagent called YeastBuster to the samples, which allows for fast extraction of native proteins from yeast without mechanical disruption and enzymatic lysis, mixed with NanoBiT substrate.


figure 4: Luciferase assay.

No correlation between IL-6 concentration and luminescence intensity was observed at any incubation time. This indicates, that the biosensor does not work as intended for the concentrations and experimental conditions of the experiment. A similar assay was performed with the IL-10 biosensor strain also developed by the UCopenhagen 2020 team (link to biobricks). Compared with the IL-10 biosensor, the amount of luminescence was between 3-10 times higher at all concentrations and incubation times.

figure 4: Luciferase assay.

This suggests that the two extracellular domains have an affinity to each other even without the presence of IL-6. This further implies that the proteins produced from BBa_K3617000 and BBa_K3617001 are localized at the same subcellular compartment(s). The high amount of luminescence may also be caused by partial degradation of BBa_K3617001 may also be partially degraded, after which the synthetic transcription factor, lexA-VP16, is released and re-localizes to the nucleus. This could be verified by integrating only the BBa_K3617001 and reporter gene into S. cerevisiae and performing an additional luciferase assay. Alternatively, one may perform a western blot with primary antibody against GFP on the strain used for the localization assays.