Difference between revisions of "Part:BBa K4160012"
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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 using a MATLAB script (Figure 6). 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-HA, the absorbance values at 405 nm were measured. From these absorbance values, the SEAP activity was calculated using a MATLAB script (Figure 6). This MATLAB script can be found on the part page of SEAP (<a href="https://parts.igem.org/Part:BBa_K147004">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/b3/BBa_K4160012_TU-Eindhoven_receptor_SEAPassay.png" width="640px" heigth=480px"> | <figure><img src="https://static.igem.org/mediawiki/parts/b/b3/BBa_K4160012_TU-Eindhoven_receptor_SEAPassay.png" width="640px" heigth=480px"> | ||
Revision as of 10:35, 12 October 2022
GEMS receptor construct containing PR3 fused to 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 PR3 (BBa_K4160004) fused to HA-tag (BBa_K1150016) as affinity domain (Figure 1).
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. In addition to ligand anti-PR3, this receptor should sense the ligand anti-HA as illustrated.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 760
Illegal XbaI site found at 2366
Illegal PstI site found at 191
Illegal PstI site found at 1054
Illegal PstI site found at 2049
Illegal PstI site found at 2210 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 760
Illegal NheI site found at 1423
Illegal PstI site found at 191
Illegal PstI site found at 1054
Illegal PstI site found at 2049
Illegal PstI site found at 2210
Illegal NotI site found at 2353 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 760
Illegal BglII site found at 1527
Illegal BglII site found at 1713
Illegal BglII site found at 1977
Illegal BamHI site found at 64
Illegal XhoI site found at 937
Illegal XhoI site found at 2360 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 760
Illegal XbaI site found at 2366
Illegal PstI site found at 191
Illegal PstI site found at 1054
Illegal PstI site found at 2049
Illegal PstI site found at 2210 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 760
Illegal XbaI site found at 2366
Illegal PstI site found at 191
Illegal PstI site found at 1054
Illegal PstI site found at 2049
Illegal PstI site found at 2210 - 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 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 an 8 amino acid (BBa_K4160013) 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.
Receptor binding
To investigate the binding of anti-PR3 and anti-HA antibodies to their specific antigen, 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 or a primary anti-PR3 and secondary antibody with Alexa Fluor 488. Flow cytometry experiments were performed using a FACS instrument (Figure 3 & 4).
Figure 3 | Flow cytometry results of PR3-HA with anti-HA staining. Flow cytometry results of the receptor construct with PR3-HA affinity domain, 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 10.3%, indicating the percentage of HEK293T cells that have bound anti-HA via their GEMS receptor construct was obtained (Figure 3).
Figure 4 shows the percentages for the bound anti-PR3 to the GEMS receptor contstruct which increased to 21.9% compared to the negative control.
Figure 4 | Flow cytometry results of receptor constructs PR3-HA with anti-PR3 staining. Flow cytometry results of the receptor construct with PR3-HA as affinity domain stained with primary anti-PR3 antibodies (blue) and subsequent anti-IgG antibodies (Alexa Fluor 488, orange). 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.
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, 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 5).
Figure 5 | 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 using a MATLAB script (Figure 6). This MATLAB script can be found on the part page of SEAP (BBa_K147004), which was contributed by the TU-Eindhoven team 2022.
Figure 6 | Antibody-induced receptor activation. HEK293T cells were transiently transfected with 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