Difference between revisions of "Part:BBa K4160009"
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| − | <p>To determine the activity of the GEMS receptor upon the addition of increasing concentrations of ligand anti-PR3, the absorbance values at 405 nm were measured. From these absorbance values, the SEAP activity was calculated using a MATLAB script (Figure 4). This MATLAB script can be found on the part page of SEAP (<a href="https://parts.igem.org/Part: | + | <p>To determine the activity of the GEMS receptor upon the addition of increasing concentrations of ligand anti-PR3, the absorbance values at 405 nm were measured. From these absorbance values, the SEAP activity was calculated using a MATLAB script (Figure 4). This MATLAB script can be found on the part page of SEAP (<a href="https://parts.igem.org/Part:BBa_I147004">BBa_K147004</a>), which was contributed by the <a href="https://2022.igem.wiki/tu-eindhoven/">TU-Eindhoven team 2022.</a></p><br> |
<figure><img src="https://static.igem.org/mediawiki/parts/b/ba/BBa_K4160009_TU-Eindhoven_receptor_SEAPassay.png" width="640px" heigth=480px"> | <figure><img src="https://static.igem.org/mediawiki/parts/b/ba/BBa_K4160009_TU-Eindhoven_receptor_SEAPassay.png" width="640px" heigth=480px"> | ||
Revision as of 10:31, 12 October 2022
GEMS receptor construct containing PR3 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 PR3 affinity domain (BBa_K4160004) (Figure 1).
This PR3 domain 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 as affinity domain. This receptor should sense the ligand anti-PR3.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 733
Illegal XbaI site found at 2339
Illegal PstI site found at 191
Illegal PstI site found at 1027
Illegal PstI site found at 2022
Illegal PstI site found at 2183 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 733
Illegal NheI site found at 1396
Illegal PstI site found at 191
Illegal PstI site found at 1027
Illegal PstI site found at 2022
Illegal PstI site found at 2183
Illegal NotI site found at 2326 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 733
Illegal BglII site found at 1500
Illegal BglII site found at 1686
Illegal BglII site found at 1950
Illegal BamHI site found at 64
Illegal XhoI site found at 910
Illegal XhoI site found at 2333 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 733
Illegal XbaI site found at 2339
Illegal PstI site found at 191
Illegal PstI site found at 1027
Illegal PstI site found at 2022
Illegal PstI site found at 2183 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 733
Illegal XbaI site found at 2339
Illegal PstI site found at 191
Illegal PstI site found at 1027
Illegal PstI site found at 2022
Illegal PstI site found at 2183 - 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 could be activated using autoantibodies, specifically anti-PR3, as a ligand. This part is a member of a library that was created. Additional parts of this library are the GEMS receptor constructs containing the PR3 affinity domain fused to EpoR via an 8 amino acid (BBa_K4160010) and a 31 amino acid (BBa_K4160011) 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 as affinity domain.
To determine the activity of the GEMS receptor upon the addition of increasing concentrations of ligand anti-PR3, the absorbance values at 405 nm were measured. From these absorbance values, the SEAP activity was calculated using a MATLAB script (Figure 4). This MATLAB script can be found on the part page of SEAP (BBa_K147004), which was contributed by the TU-Eindhoven team 2022.
Figure 4 | Antibody-induced receptor activation. HEK293T cells were transiently transfected with 0_PR3, pLS13, and pLS15, and subsequently induced with a titration of anti-PR3 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 as affinity domain was unsuccessful. Antibody-induced activation of the GEMS receptor constructs remains challenging but is actively under investigation.
References