Difference between revisions of "Part:BBa K4160015"
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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 HA (<a href="https://parts.igem.org/Part:BBa_K1150016">BBa_K1150016</a>) as affinity domain (Figure 1) containing a linker of 8 amino acids.<br> The DNA sequence of this part was used as a template for mRNA</p><br> | + | <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 HA (<a href="https://parts.igem.org/Part:BBa_K1150016">BBa_K1150016</a>) as affinity domain (Figure 1) containing a linker of 8 amino acids.<br> The DNA sequence of this part was used as a template for mRNA.</p><br> |
<p>A 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> | <p>A 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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<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> | <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> | ||
| − | <p><a href="https://2022.igem.wiki/tu-eindhoven/">The TU-Eindhoven team 2022</a> developed this part to investigate whether the GEMS receptor would be expressed properly on the cellular membrane of <i>HEK293T</i> cells. This part was expressed using mRNA</p><br> | + | <p><a href="https://2022.igem.wiki/tu-eindhoven/">The TU-Eindhoven team 2022</a> developed this part to investigate whether the GEMS receptor would be expressed properly on the cellular membrane of <i>HEK293T</i> cells. This part was expressed using mRNA.</p><br> |
<h2>Characterization</h2> | <h2>Characterization</h2> | ||
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| − | <p><b>Figure 3 | Flow cytometry results of 8_HA with anti-HA staining after 24 and 48 hours. A & B</b> Flow cytometry results of the receptor construct with HA-tag affinity domain containing a linker of 8 amino acids, stained with anti-HA (Alexa Fluor 488) antibodies, 24 and 48 hours after mRNA transfection. Green shading and percentages depict the number of cells with a higher fluorescence intensity in the Alexa Fluor 488 channel than the negative control. Percentages and cell counts were determined after gating. Data was acquired using the BD FACSymphony A3 (BD Biosciences) and visualized using FlowJo software.</p> | + | <p><b>Figure 3 | Flow cytometry results of 8_HA with anti-HA staining after 24 and 48 hours. A</b> & <b>B</b> Flow cytometry results of the receptor construct with HA-tag affinity domain containing a linker of 8 amino acids, stained with anti-HA (Alexa Fluor 488) antibodies, 24 and 48 hours after mRNA transfection. Green shading and percentages depict the number of cells with a higher fluorescence intensity in the Alexa Fluor 488 channel than the negative control. Percentages and cell counts were determined after gating. Data was acquired using the BD FACSymphony A3 (BD Biosciences) and visualized using FlowJo software.</p> |
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</figure><br> | </figure><br> | ||
Latest revision as of 14:17, 12 October 2022
GEMS receptor construct containing HA-tag as affinity domain
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 HA (BBa_K1150016) as affinity domain (Figure 1) containing a linker of 8 amino acids.
The DNA sequence of this part was used as a template for mRNA.
A 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 HA-tag as affinity domain. The HA-tag was fused to the GEMS receptor via a linker of 8 amino acids. This receptor should sense the ligand anti-HA.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 97
Illegal XbaI site found at 1727
Illegal PstI site found at 415
Illegal PstI site found at 1410
Illegal PstI site found at 1571 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 97
Illegal NheI site found at 784
Illegal PstI site found at 415
Illegal PstI site found at 1410
Illegal PstI site found at 1571
Illegal NotI site found at 1714 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 97
Illegal BglII site found at 888
Illegal BglII site found at 1074
Illegal BglII site found at 1338
Illegal BamHI site found at 64
Illegal XhoI site found at 298
Illegal XhoI site found at 1721 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 97
Illegal XbaI site found at 1727
Illegal PstI site found at 415
Illegal PstI site found at 1410
Illegal PstI site found at 1571 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 97
Illegal XbaI site found at 1727
Illegal PstI site found at 415
Illegal PstI site found at 1410
Illegal PstI site found at 1571 - 1000COMPATIBLE 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 TU-Eindhoven team 2022 developed this part to investigate whether the GEMS receptor would be expressed properly on the cellular membrane of HEK293T cells. This part was expressed using mRNA.
Characterization
To investigate the binding of anti-HA antibodies to the HA-tag, an initial flow cytometry experiment was conducted. The mRNA encoding for the GEMS receptor construct containing HA-tag as affinity domain was transfected into HEK293T cells. Since the optimal expression of mRNA and the receptor induction was hard to estimate beforehand, incubation steps of 24 and 48 hours were performed. Subsequently, the HEK293T cells were collected and stained with a primary anti-HA antibody with Alexa Fluor 488. Flow cytometry experiments were performed using a FACS instrument (Figure 3).
Figure 3 | Flow cytometry results of 8_HA with anti-HA staining after 24 and 48 hours. A & B Flow cytometry results of the receptor construct with HA-tag affinity domain containing a linker of 8 amino acids, stained with anti-HA (Alexa Fluor 488) antibodies, 24 and 48 hours after mRNA transfection. Green shading and percentages depict the number of cells with a higher fluorescence intensity in the Alexa Fluor 488 channel than the negative control. Percentages and cell counts were determined after gating. Data was acquired using the BD FACSymphony A3 (BD Biosciences) and visualized using FlowJo software.
The green shading and percentages shown in the graphs depict the number of cells with a higher fluorescence intensity in the Alexa Fluor 488 channel than the negative control. A percentage of 11.6% and 9,7% for incubation after 24 and 48 hours, respectively, indicating the percentage of HEK293T cells that have bound anti-HA via their GEMS receptor construct was obtained (Figure 3).
This initial experiment demonstrated that successful expression of the receptor was obtained since an increase in binding between both antibodies and the GEMS receptor construct compared to the negative control was seen. Due to limited time, no optimizations could be done. However, these initial results are promising, as antibody binding to the affinity domain of the GEMS receptor construct was obtained.
References