Difference between revisions of "Part:BBa K4160014"

 
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This composite part combines BBa_K4160000, BBa_K4160001, BBa_K4160002 BBa_K4160004, BBa_K1150016, and BBa_K2217015 containing a linker of 31 amino acids.
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<p>This composite part encodes for a Generalized Extracellular Molecule Sensor (GEMS) receptor construct. This part was developed by replacing the RR120 VHH affinity domain of <a href="https://parts.igem.org/Part:BBa_K4160008">BBa_K4160008</a> with a PR3 (<a href="https://parts.igem.org/Part:BBa_K4160004">BBa_K4160004</a>)  fused to HA-tag (<a href="https://parts.igem.org/Part:BBa_K1150016">BBa_K1150016</a>)  as affinity domain (Figure 1) containing a linker of 31 amino acids.</p><br>
  
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<!-- Add more about the biology of this part here
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<p>This PR3 domain with HA-tag is fused to the erythropoietin receptor (EpoR) (<a href="https://parts.igem.org/Part:BBa_K4160001">BBa_K4160001</a>), a transmembrane receptor that forms the foundation of the GEMS receptor. At the intracellular side of the EpoR, the intracellular signal transduction domain IL-6RB (<a href="https://parts.igem.org/Part:BBa_K4160002">BBa_K4160002</a>) is attached. Sensing and binding of ligand anti-PR3 to the affinity domain should induce dimerization of the EpoR. As a result, the IL-6RB domain should activate downstream signaling of the Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway. In this part, an Igκ secretion signal (<a href="https://parts.igem.org/Part:BBa_K4160000">BBa_K4160000</a>) is incorporated. This signal localizes the GEMS receptor to the membrane of mammalian cells. Furthermore, at the C-terminus of the part, a bovine growth Hormone polyadenylation (bGH poly A) signal is located which medicates efficient transcription termination and polyadenylation.<sup>1</sup></p><br>
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===Usage and Biology===
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<figure><img src="https://static.igem.org/mediawiki/parts/a/a8/BBa_K4160014_TU-Eindhoven_receptor_HAPR3_31aa.png" width="400px" heigth=320px">
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<p><b>Figure 1 | |GEMS receptor construct containing PR3 fused to HA-tag as affinity domain.</b> PR3 with HA-tag was fused to the GEMS receptor via a linker of 31 amino acids. In addition to ligand anti-PR3, this receptor should sense the ligand anti-HA as illustrated.</p>
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'><h3>Sequence and Features</h3></span>
 
<partinfo>BBa_K4160014 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4160014 SequenceAndFeatures</partinfo>
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<br>
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<h2>Usage & biology</h2>
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<p>This GEMS receptor construct is based on the GEMS system that is developed by Scheller et al., 2018.<sup>2</sup> The authors developed this highly modular synthetic receptor construct that allows for the coupling of an extracellular input to an intracellular signaling pathway.<sup>2</sup> The modularity of this receptor allows the designing of GEMS platforms that sense and respond to a wide variety of extracellular molecules.<sup>2</sup></p><br>
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<p>The success of the designed library consisting GEMS receptors containing PR3 as affinity domain (BBa_K4160009, BBa_K4160010 & BBa_K4160011) of the<a href="https://2022.igem.wiki/tu-eindhoven/">TU-Eindhoven team 2022</a> is dependent on the correct folding of the truncated PR3. Incorrect folding of PR3 prevents the anti-PR3 antibody from binding to the affinity domain and hence prevents activation of the GEMS receptor. Therefore, a library of GEMS receptor constructs containing a HA-tag at the C-terminus of PR3 was designed. With these receptor constructs, it could not only be investigated whether the GEMS receptor could be activated by antibodies, in general, using anti-HA but also by disease-related antibodies using anti-PR3.</p><br>
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<p>This part is a member of a library that was created. Additional parts of this library are the GEMS receptor constructs containing the PR3 fused to HA-tag as affinity domain fused to EpoR with no (<a href="https://parts.igem.org/Part:BBa_K4160012">BBa_K4160012</a>) and a 8 amino acid (<a href="https://parts.igem.org/Part:BBa_K4160013">BBa_K4160013</a>) linker.</p><br>
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<p>This composite part was used in combination with the transcription factor Signal Transducer and Activator of transcription 3 (STAT3) (<a href="https://parts.igem.org/Part:BBa_K4160005">BBa_K4160005</a>) and the part that encodes STAT-induced SEAP (<a href="https://parts.igem.org/Part:BBa_K4160016">BBa_K4160016</a>). This part was expressed using a pLeo619-P<sub>sv40</sub> mammalian expression vector (GenBank accession no. MG437012).<sup>3</sup></p><br>
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<h2>Characterization</h2>
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<p>Unfortunately, cloning of PR3 fused to HA-tag containing a 31 amino acid linker into the pLeo619-P<sub>sv40</sub> expression vector was unsuccessful. Therefore, no colonies were grown after transfection and no characterization experiments could be performed.</p><br><br>
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<h2>References</h2>
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<p><small>
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<ol>
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<li>Wang XY, Du QJ, Zhang WL, et al. Enhanced Transgene Expression by Optimization of Poly A in Transfected CHO Cells. Front Bioeng Biotechnol. 2022;10. doi:10.3389/FBIOE.2022.722722/FULL</li>
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<li>Scheller L, Strittmatter T, Fuchs D, Bojar D, Fussenegger M. Generalized extracellular molecule sensor platform for programming cellular behavior. Nat Chem Biol. Published online 2018. doi:10.1038/s41589-018-0046-z</li>
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<li>Expression vector pLeo619, complete sequence - Nucleotide - NCBI. Accessed September 8, 2022. https://www.ncbi.nlm.nih.gov/nuccore/MG437012</li>
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</ol>
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</small></p><br><br>
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</body>
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</html>
  
  

Revision as of 23:15, 11 October 2022


GEMS receptor construct containing PR3 fused to HA-tag as affinity domain with 31 amino acid linker

This composite part encodes for a Generalized Extracellular Molecule Sensor (GEMS) receptor construct. This part was developed by replacing the RR120 VHH affinity domain of BBa_K4160008 with a PR3 (BBa_K4160004) fused to HA-tag (BBa_K1150016) as affinity domain (Figure 1) containing a linker of 31 amino acids.


This PR3 domain with HA-tag is fused to the erythropoietin receptor (EpoR) (BBa_K4160001), a transmembrane receptor that forms the foundation of the GEMS receptor. At the intracellular side of the EpoR, the intracellular signal transduction domain IL-6RB (BBa_K4160002) is attached. Sensing and binding of ligand anti-PR3 to the affinity domain should induce dimerization of the EpoR. As a result, the IL-6RB domain should activate downstream signaling of the Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway. In this part, an Igκ secretion signal (BBa_K4160000) is incorporated. This signal localizes the GEMS receptor to the membrane of mammalian cells. Furthermore, at the C-terminus of the part, a bovine growth Hormone polyadenylation (bGH poly A) signal is located which medicates efficient transcription termination and polyadenylation.1


Figure 1 | |GEMS receptor construct containing PR3 fused to HA-tag as affinity domain. PR3 with HA-tag was fused to the GEMS receptor via a linker of 31 amino acids. In addition to ligand anti-PR3, this receptor should sense the ligand anti-HA as illustrated.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 760
    Illegal XbaI site found at 2441
    Illegal PstI site found at 191
    Illegal PstI site found at 1129
    Illegal PstI site found at 2124
    Illegal PstI site found at 2285
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 760
    Illegal NheI site found at 1498
    Illegal PstI site found at 191
    Illegal PstI site found at 1129
    Illegal PstI site found at 2124
    Illegal PstI site found at 2285
    Illegal NotI site found at 2428
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 760
    Illegal BglII site found at 1602
    Illegal BglII site found at 1788
    Illegal BglII site found at 2052
    Illegal BamHI site found at 64
    Illegal XhoI site found at 1012
    Illegal XhoI site found at 2435
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 760
    Illegal XbaI site found at 2441
    Illegal PstI site found at 191
    Illegal PstI site found at 1129
    Illegal PstI site found at 2124
    Illegal PstI site found at 2285
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 760
    Illegal XbaI site found at 2441
    Illegal PstI site found at 191
    Illegal PstI site found at 1129
    Illegal PstI site found at 2124
    Illegal PstI site found at 2285
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage & biology

This GEMS receptor construct is based on the GEMS system that is developed by Scheller et al., 2018.2 The authors developed this highly modular synthetic receptor construct that allows for the coupling of an extracellular input to an intracellular signaling pathway.2 The modularity of this receptor allows the designing of GEMS platforms that sense and respond to a wide variety of extracellular molecules.2


The success of the designed library consisting GEMS receptors containing PR3 as affinity domain (BBa_K4160009, BBa_K4160010 & BBa_K4160011) of theTU-Eindhoven team 2022 is dependent on the correct folding of the truncated PR3. Incorrect folding of PR3 prevents the anti-PR3 antibody from binding to the affinity domain and hence prevents activation of the GEMS receptor. Therefore, a library of GEMS receptor constructs containing a HA-tag at the C-terminus of PR3 was designed. With these receptor constructs, it could not only be investigated whether the GEMS receptor could be activated by antibodies, in general, using anti-HA but also by disease-related antibodies using anti-PR3.


This part is a member of a library that was created. Additional parts of this library are the GEMS receptor constructs containing the PR3 fused to HA-tag as affinity domain fused to EpoR with no (BBa_K4160012) and a 8 amino acid (BBa_K4160013) linker.


This composite part was used in combination with the transcription factor Signal Transducer and Activator of transcription 3 (STAT3) (BBa_K4160005) and the part that encodes STAT-induced SEAP (BBa_K4160016). This part was expressed using a pLeo619-Psv40 mammalian expression vector (GenBank accession no. MG437012).3


Characterization

Unfortunately, cloning of PR3 fused to HA-tag containing a 31 amino acid linker into the pLeo619-Psv40 expression vector was unsuccessful. Therefore, no colonies were grown after transfection and no characterization experiments could be performed.



References

  1. Wang XY, Du QJ, Zhang WL, et al. Enhanced Transgene Expression by Optimization of Poly A in Transfected CHO Cells. Front Bioeng Biotechnol. 2022;10. doi:10.3389/FBIOE.2022.722722/FULL
  2. Scheller L, Strittmatter T, Fuchs D, Bojar D, Fussenegger M. Generalized extracellular molecule sensor platform for programming cellular behavior. Nat Chem Biol. Published online 2018. doi:10.1038/s41589-018-0046-z
  3. Expression vector pLeo619, complete sequence - Nucleotide - NCBI. Accessed September 8, 2022. https://www.ncbi.nlm.nih.gov/nuccore/MG437012