Difference between revisions of "Part:BBa K3617002"
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Superfolding green flourescent protein (sfGFP) was linked C-terminally to the protein and the cells where observed with <span>confocal flourescence microscopy.</span>
 
Superfolding green flourescent protein (sfGFP) was linked C-terminally to the protein and the cells where observed with <span>confocal flourescence microscopy.</span>
  
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[[Image:T--UCopenhagen--results-localization25 1.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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[[Image:T--UCopenhagen--results-localization25 1.jpg|700px|thumb|center|<p align="justify"> '''figure 2a: Pictures were taken with a 150 &mu;m pinhole. The image shows localization of the protein at the membrane, and a few inclusion bodies'''</p>]]
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[[Image:T--UCopenhagen--results-localization25 2flour.jpg|700px|thumb|center|<p align="justify"> '''figure 3b: Pictures were taken with a 150 &mu;m pinhole. Here the inclusion bodies are also evident in the brightfield image.'''</p>]]
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[[Image:T--UCopenhagen--results-localization25 2flour.jpg|700px|thumb|center|<p align="justify"> '''figure 2b: Pictures were taken with a 150 &mu;m pinhole. The image shows localization to the membrane as well as the endoplasmatic reticulum'''</p>]]
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[[Image:T--UCopenhagen--results-localization25 3flour.jpg|700px|thumb|center|<p align="justify"> '''figure 3c: Pictures were taken with a 150 &mu;m pinhole. Flourescence in inclusion bodies and very faintly at membrane and around nucleus'''</p>]]
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[[Image:T--UCopenhagen--results-localization25 3flour.jpg|700px|thumb|center|<p align="justify"> '''figure 2c: Pictures were taken with a 150 &mu;m pinhole. Flourescence here is mainly found around nucleus and in a reticulum throughout the cell which might indicate localisation to the ER'''</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.
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Most of the cells had flourescence at the plasma membrane an around the nucleus in what looks to be the endoplasmatic reticulum. The localization to other subcellular compartments than the plasma membrane, ie. endoplasmatic reticulum, might be a result of a very high expression rate as the biobrick was put under the constitutive promoter pCCW12 and this might "clog" the secretory pathway.
  
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<h2>Biosensor assays</h2>
 
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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.
 
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.
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Revision as of 17:38, 22 October 2020


Interleukin-10 receptor type 1 - C-ubiquitin-LexA-VP16

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 refered to as Wsc1 while the gene is called SLG1 what do we call it?) receptor in S. Cerevisiae.
  • The first and second domain (aa 22-235) of human interleukin-10 receptor subunit α (IL-10RI).
  • The transmembrane domain from Wsc1
  • C-terminal part of a split version of ubiquitin. This means the last 42 amino acids (aa 35-76) of the ubiquitin protein. When binding to the N-terminal part of the split-protein, an endogenous deubiquitinase can cleave the polypeptide chain just C-terminally of the C-terminal part of the split protein.
  • Between the IL-10R1 domains and the transmembrane domain we added a flexible 2XGGGGS linker (cite: PMID: 23026637) and between the transmembrane domain and the C-terminal split ubiquitin domain we added two basic amino acids; KR, and the 2XGGGGS linker again.

Expected function of the protein:

This part is meant to function as a IL-10 receptor together with BBaK3617003. Upon complementation of the split ubiquitin halves, the synthetic transcription factor lexA-VP16 which the relocates to the nucleus and mediates transcription of genes controlled by promoters with Lexo sites.

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 Wsc1 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 interleukin-10 receptor subunit α domains are mediating the binding of the receptor to interleukin-10 - as seen in crystal structures of the receptor (see fig. 1). This biobrick is intended to work together with BBaK3617003which has the outer two domains of the IL-10 receptor subunit β extracellularly and the N-terminal part of split ubiquitin intracellularly. We hypothesized that BBa_K3617002 (this biobrick) and BBaK3617003would both localize to the same membrane but that they would be dissociated in the absence of interleukin-10. In the presence of interleukin-10, we imagined that the extracellular domains of the two parts; IL-10Rα and IL-10Rβ, would associate into a heterotrimer consisting of IL-10, IL-10Rα and IL-10R&beta. 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.

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.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 628
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1269
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Confocal flourescence microscopy

Superfolding green flourescent protein (sfGFP) was linked C-terminally to the protein and the cells where observed with confocal flourescence microscopy.

figure 2a: Pictures were taken with a 150 μm pinhole. The image shows localization of the protein at the membrane, and a few inclusion bodies

figure 2b: Pictures were taken with a 150 μm pinhole. The image shows localization to the membrane as well as the endoplasmatic reticulum

figure 2c: Pictures were taken with a 150 μm pinhole. Flourescence here is mainly found around nucleus and in a reticulum throughout the cell which might indicate localisation to the ER


Most of the cells had flourescence at the plasma membrane an around the nucleus in what looks to be the endoplasmatic reticulum. The localization to other subcellular compartments than the plasma membrane, ie. endoplasmatic reticulum, might be a result of a very high expression rate as the biobrick was put under the constitutive promoter pCCW12 and this might "clog" the secretory pathway.