Difference between revisions of "Part:BBa K4160013"

 
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<partinfo>BBa_K4160013 short</partinfo>
 
<partinfo>BBa_K4160013 short</partinfo>
  
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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 8 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 8 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/2/2c/BBa_K4160013_TU-Eindhoven_receptor_HAPR3_8aa.png" width="400px" heigth=320px">
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<figcaption>
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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 8 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'><h2>Sequence and Features</h2></span>
 
<partinfo>BBa_K4160013 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4160013 SequenceAndFeatures</partinfo>
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<html>
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<body>
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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 31 amino acid (<a href="https://parts.igem.org/Part:BBa_K4160014">BBa_K4160014</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>This composite part was successfully transformed into TOP10 Chemically Competent <i>E. coli</i> cells (Figure 2). To multiply the amount of plasmid, colonies were picked and small cultures were made. After this, the plasmids were purified with a <a href="https://2022.igem.wiki/tu-eindhoven/notebook">miniprep kit</a>.</p><br>
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<figure><img src="https://static.igem.org/mediawiki/parts/6/65/BBa_K4160013_TU-Eindhoven_agarplate.png" width="350px" heigth=300px">
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<figcaption>
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<p><b>Figure 2 | Agar plate transfected with pLeo619-P<sub>sv40</sub> in TOP10 Chemically Competent <i>E. coli</i> cells.</b> This plasmid contained the GEMS receptor construct containing PR3 fused to HA-tag as affinity domain containing a linker of 8 amino acids.</p>
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</figcaption>
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</figure><br>
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<h3>Receptor binding</h3>
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<p>To investigate the binding of anti-HA antibodies to the HA-tag, an initial flow cytometry experiment was conducted. the pLeo619-P<sub>sv40</sub> plasmid was transfected into <i>HEK293T</i> cells, followed by an incubation and receptor induction step of minimally 40 hours. Subsequently, the <i>HEK293T</i> 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).</p><br>
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<figure><img src="https://static.igem.org/mediawiki/parts/3/37/BBa_K4160013_TU-Eindhoven_receptor_FACS_antiHA.png" width="480px" heigth=320px">
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<figcaption>
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<p><b>Figure 3 | Flow cytometry results of PR3-HA with anti-HA staining.</b> Flow cytometry results of the receptor construct with PR3-HA affinity domain containing a linker of 8 amino acids, stained with anti-HA (Alexa Fluor 488) antibodies. 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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</figcaption>
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</figure><br>
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<p>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 11.5%, indicating the percentage of <i>HEK293T</i> cells that have bound anti-HA via their GEMS receptor construct was obtained (Figure 3).</p><br>
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<p>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.</p><br>
 +
 +
<h3>Receptor activation</h3>
 +
<p>To investigate antibody-induced activation of the GEMS receptor construct containing PR3 fused to anti-HA as affinity domain with an 8 amino acid linker, the pLeo619-P<sub>sv40</sub> plasmids were transfected into <i>HEK293T</i> cells, together with the pLS15 plasmid that encodes for STAT3 (<a href="https://parts.igem.org/Part:BBa_K4160005">BBa_K4160005</a>) and the pLS13 plasmid that encodes for STAT-induced SEAP (<a href="https://parts.igem.org/Part:BBa_K4160016">BBa_K4160016</a>). Subsequently, ligand titration with anti-HA on the transfected cells was performed, followed by an incubation and receptor induction step of minimally 40 hours (Figure 4).</p><br>
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<figure><img src="https://static.igem.org/mediawiki/parts/c/cc/BBa_K4160012_TU-Eindhoven_receptor_SEAPassay_overview.png" width="320px" heigth=220px">
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<figcaption>
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<p><b>Figure 4 | Anti-HA induced receptor activation.</b> Schematic representation of GEMS receptor induction by anti-HA, resulting in SEAP secretion.</p>
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</figcaption>
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</figure><br>
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<p>To determine the activity of the GEMS receptor upon the addition of increasing concentrations of ligand anti-HA, the absorbance values at 405 nm were measured. From these absorbance values, the SEAP activity was calculated (Figure 5). This MATLAB script can be found on the part page of SEAP (<a href="https://parts.igem.org/Part:BBa_K4160004">BBa_K147004</a>), which was contributed by the <a href="https://2022.igem.wiki/tu-eindhoven/">TU-Eindhoven team 2022</a>.</p><br>
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<figure><img src="https://static.igem.org/mediawiki/parts/8/8a/BBa_K4160013_TU-Eindhoven_receptor_SEAPassay.png" width="640px" heigth=480px">
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<figcaption>
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<p><b>Figure 5 | Antibody-induced receptor activation.</b> <i>HEK293T</i> cells were transiently transfected with 8_PR3-HA, pLS13, and pLS15, and subsequently induced with a titration of anti-HA antibodies (OriGene, cat. Nr. TA807348). Cells were incubated for minimally 40 h. All experiments were performed in biological triplicates, for which 5 µL of cell medium (incubation for 30 minutes at 65 °C) was used. Measurements were taken every 30 seconds for 1 hour at 405 nm at RT (25 °C). Data was processed by the SEAP MATLAB script, which calculates the SEAP activity using the measured absorbance at 405 nm. Bars represent mean values, overlayed individual data points are represented as circles (for n=3 biologically independent samples). The inset figure shows a zoomed-in view of the same data.</p>
 +
</figcaption>
 +
</figure><br>
 +
 +
<p>Unfortunately, the activation of the GEMS receptor construct containing PR3 fused to HA-tag as affinity domain was unsuccessful. Antibody-induced activation of the GEMS receptor constructs remains challenging but is actively under investigation.</p><br><br>
 +
 +
<h2>References</h2>
 +
<p><small>
 +
<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>
 +
<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>
 +
<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 23:11, 11 October 2022


GEMS receptor construct containing PR3 fused to HA-tag as affinity domain with 8 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 8 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 8 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 2390
    Illegal PstI site found at 191
    Illegal PstI site found at 1078
    Illegal PstI site found at 2073
    Illegal PstI site found at 2234
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 760
    Illegal NheI site found at 1447
    Illegal PstI site found at 191
    Illegal PstI site found at 1078
    Illegal PstI site found at 2073
    Illegal PstI site found at 2234
    Illegal NotI site found at 2377
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 760
    Illegal BglII site found at 1551
    Illegal BglII site found at 1737
    Illegal BglII site found at 2001
    Illegal BamHI site found at 64
    Illegal XhoI site found at 961
    Illegal XhoI site found at 2384
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 760
    Illegal XbaI site found at 2390
    Illegal PstI site found at 191
    Illegal PstI site found at 1078
    Illegal PstI site found at 2073
    Illegal PstI site found at 2234
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 760
    Illegal XbaI site found at 2390
    Illegal PstI site found at 191
    Illegal PstI site found at 1078
    Illegal PstI site found at 2073
    Illegal PstI site found at 2234
  • 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 31 amino acid (BBa_K4160014) 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

This composite part was successfully transformed into TOP10 Chemically Competent E. coli cells (Figure 2). To multiply the amount of plasmid, colonies were picked and small cultures were made. After this, the plasmids were purified with a miniprep kit.


Figure 2 | Agar plate transfected with pLeo619-Psv40 in TOP10 Chemically Competent E. coli cells. This plasmid contained the GEMS receptor construct containing PR3 fused to HA-tag as affinity domain containing a linker of 8 amino acids.


Receptor binding

To investigate the binding of anti-HA antibodies to the HA-tag, an initial flow cytometry experiment was conducted. the pLeo619-Psv40 plasmid was transfected into HEK293T cells, followed by an incubation and receptor induction step of minimally 40 hours. 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 PR3-HA with anti-HA staining. Flow cytometry results of the receptor construct with PR3-HA affinity domain containing a linker of 8 amino acids, stained with anti-HA (Alexa Fluor 488) antibodies. 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 11.5%, 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.


Receptor activation

To investigate antibody-induced activation of the GEMS receptor construct containing PR3 fused to anti-HA as affinity domain with an 8 amino acid linker, the pLeo619-Psv40 plasmids were transfected into HEK293T cells, together with the pLS15 plasmid that encodes for STAT3 (BBa_K4160005) and the pLS13 plasmid that encodes for STAT-induced SEAP (BBa_K4160016). Subsequently, ligand titration with anti-HA on the transfected cells was performed, followed by an incubation and receptor induction step of minimally 40 hours (Figure 4).


Figure 4 | Anti-HA induced receptor activation. Schematic representation of GEMS receptor induction by anti-HA, resulting in SEAP secretion.


To determine the activity of the GEMS receptor upon the addition of increasing concentrations of ligand anti-HA, the absorbance values at 405 nm were measured. From these absorbance values, the SEAP activity was calculated (Figure 5). This MATLAB script can be found on the part page of SEAP (BBa_K147004), which was contributed by the TU-Eindhoven team 2022.


Figure 5 | Antibody-induced receptor activation. HEK293T cells were transiently transfected with 8_PR3-HA, pLS13, and pLS15, and subsequently induced with a titration of anti-HA antibodies (OriGene, cat. Nr. TA807348). Cells were incubated for minimally 40 h. All experiments were performed in biological triplicates, for which 5 µL of cell medium (incubation for 30 minutes at 65 °C) was used. Measurements were taken every 30 seconds for 1 hour at 405 nm at RT (25 °C). Data was processed by the SEAP MATLAB script, which calculates the SEAP activity using the measured absorbance at 405 nm. Bars represent mean values, overlayed individual data points are represented as circles (for n=3 biologically independent samples). The inset figure shows a zoomed-in view of the same data.


Unfortunately, the activation of the GEMS receptor construct containing PR3 fused to HA-tag as affinity domain was unsuccessful. Antibody-induced activation of the GEMS receptor constructs remains challenging but is actively under investigation.



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