Difference between revisions of "Part:BBa K4160010"
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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/8/8d/BBa_K4160010_TU-Eindhoven_receptor_SEAPassay.png" width="640px" heigth=480px"> | <figure><img src="https://static.igem.org/mediawiki/parts/8/8d/BBa_K4160010_TU-Eindhoven_receptor_SEAPassay.png" width="640px" heigth=480px"> | ||
Revision as of 10:30, 12 October 2022
GEMS receptor construct containing PR3 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 affinity domain (BBa_K4160004) containing a linker of 8 amino acids (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. PR3 was fused to the GEMS receptor via a linker of 8 amino acids. 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 2363
Illegal PstI site found at 191
Illegal PstI site found at 1051
Illegal PstI site found at 2046
Illegal PstI site found at 2207 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 733
Illegal NheI site found at 1420
Illegal PstI site found at 191
Illegal PstI site found at 1051
Illegal PstI site found at 2046
Illegal PstI site found at 2207
Illegal NotI site found at 2350 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 733
Illegal BglII site found at 1524
Illegal BglII site found at 1710
Illegal BglII site found at 1974
Illegal BamHI site found at 64
Illegal XhoI site found at 934
Illegal XhoI site found at 2357 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 733
Illegal XbaI site found at 2363
Illegal PstI site found at 191
Illegal PstI site found at 1051
Illegal PstI site found at 2046
Illegal PstI site found at 2207 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 733
Illegal XbaI site found at 2363
Illegal PstI site found at 191
Illegal PstI site found at 1051
Illegal PstI site found at 2046
Illegal PstI site found at 2207 - 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 with no (BBa_K4160009) 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 DH5α 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 DH5α Chemically Competent E. coli cells. This plasmid contained the GEMS receptor construct containing PR3 as affinity domain.
To investigate antibody-induced activation of the GEMS receptor construct containing PR3 as affinity domain, 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 on the transfected cells was performed, followed by an incubation and receptor induction step of minimally 40 hours (Figure 3).
Figure 3 | Anti-PR3 induced receptor activation. Schematic representation of GEMS receptor induction by anti-PR3, resulting in SEAP secretion.
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 8_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 with an 8 amino acid linker was unsuccessful. Antibody-induced activation of the GEMS receptor constructs remains challenging but is actively under investigation.
References