Difference between revisions of "Part:BBa K4160008"
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| − | <p>To determine receptor activation of the GEMS receptor construct containing RR120 VHH as affinity domain, the pLeo619-P<sub>sv40</sub> | + | <p>To determine receptor activation of the GEMS receptor construct containing RR120 VHH as affinity domain, the pLeo619-P<sub>sv40</sub> plasmid was transfected into HEK293T 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_K41600016">BBa_K4160016</a>). Subsequently, ligand titration on the transfected cells was performed, followed by an incubation and receptor induction step of minimally 40 hours (Figure 3A). To determine the activity of the GEMS receptor upon the addition of increasing concentrations of ligand RR120, the absorbance values at 405 nm were measured. From these absorbance values, the SEAP activity was calculated (Figure 3B). This MATLAB script can be found on the part page of SEAP (<a href="https://parts.igem.org/Part:BBa_K1470004">BBa_K1470004</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/0/0c/BBa_K4160008_TU-Eindhoven_SEAPresults.png" width="640px" heigth=480px"> | <figure><img src="https://static.igem.org/mediawiki/parts/0/0c/BBa_K4160008_TU-Eindhoven_SEAPresults.png" width="640px" heigth=480px"> | ||
Revision as of 21:50, 11 October 2022
GEMS receptor construct containing RR120 VHH as affinity domain
This composite part encodes for a Generalized Extracellular Molecule Sensor (GEMS) receptor construct that contains an RR120 VHH affinity domain (BBa_K4160003) (Figure 1). This 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 azo dye RR120 to the affinity domain induces dimerization of the EpoR. As a result, the IL-6RB domain activates 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) (BBa_K2217015) signal is located which medicates efficient transcription termination and polyadenylation.1
Figure 1 | GEMS receptor construct containing RR120 VHH as affinity domain. This receptor can sense the ligand azo dye RR120.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 454
Illegal XbaI site found at 2060
Illegal PstI site found at 748
Illegal PstI site found at 1743
Illegal PstI site found at 1904 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 454
Illegal NheI site found at 1117
Illegal PstI site found at 748
Illegal PstI site found at 1743
Illegal PstI site found at 1904
Illegal NotI site found at 2047 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 454
Illegal BglII site found at 1221
Illegal BglII site found at 1407
Illegal BglII site found at 1671
Illegal BamHI site found at 64
Illegal XhoI site found at 631
Illegal XhoI site found at 2054 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 454
Illegal XbaI site found at 2060
Illegal PstI site found at 748
Illegal PstI site found at 1743
Illegal PstI site found at 1904 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 454
Illegal XbaI site found at 2060
Illegal PstI site found at 748
Illegal PstI site found at 1743
Illegal PstI site found at 1904 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
The GEMS receptor construct is an example of 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 to design GEMS platforms that sense and respond to a wide variety of extracellular molecules.2
The TU-Eindhoven team 2022 used this composite part in combination with the transcription factor Signal Transducer and Activator of transcription 3 (STAT3) (BBa_K4160005) and parts that encode for STAT-induced proteins, including STAT-induced SEAP (BBa_K4160016) and STAT-induced IL-10 (BBa_K4160017). This part was expressed using a pLeo619-Psv40 mammalian expression vector (GenBank accession no. MG437012).3
The GEMS receptor construct containing RR120 VHH as an affinity domain is a readily reproducible part. In addition, it is highly modular, as the affinity domain successfully was replaced by other affinity domains (BBa_K4160009 & BBa_K4160012). Hence, this composite part has much potential for therapeutics, as it provides a valuable tool for the development of cell-based therapies.2 We encourage future iGEM teams to recognize its potential and include this in their projects.
Characterization
This composite part was successfully transformed into DH5α 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 competent E.coli cells. This plasmid contained the GEMS receptor construct containing RR120 VHH as affinity domain.
To determine receptor activation of the GEMS receptor construct containing RR120 VHH as affinity domain, the pLeo619-Psv40 plasmid was 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 3A). To determine the activity of the GEMS receptor upon the addition of increasing concentrations of ligand RR120, the absorbance values at 405 nm were measured. From these absorbance values, the SEAP activity was calculated (Figure 3B). This MATLAB script can be found on the part page of SEAP (BBa_K1470004), which was contributed by the TU-Eindhoven team 2022.
Figure 3 | Receptor transfection and activation analysis. A Schematic representation of GEMS receptor induction by RR120, resulting in SEAP secretion. B HEK293T cells were transiently transfected with pLEO619, pLS13, and pLS15, and subsequently induced with a titration of RR120. 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). Each bar shows its fold increase of SEAP activity, compared to the 0 ng/mL condition.
The GEMS receptor was successfully transfected into HEK293T cells since receptor activation was only obtained for the conditions treated with different concentrations of RR120. Receptor activations were depicted by increased SEAP activity for the conditions that were induced by RR120 when compared to the uninduced condition. A 32-fold and 34-fold change in SEAP activity is seen between the uninduced and induced (100 and 300 ng/ml) conditions, respectively. Moreover, the maximum receptor activation for the measured ligand concentrations is seen for 300 ng/ml RR120.
After receptor activation was successfully obtained, we continued with exchanging the SEAP gene for the IL-10 gene that enables expression of a therapeutically relevant protein. Due to the modularity of the GEMS system, this alteration was performed easily. IL-10 (BBa_K4160007) was cloned into the pLS13 plasmid to obtain STAT-induced IL-10 (BBa_K4160017). Together with pLeo619-Psv40 and pLS15, this pLS13 plasmid was transfected into HEK293T cells. Subsequently, the cell samples were treated with 300 ng/ml, since this concentration resulted in the highest receptor activation as described above (Figure 4).
Figure 4 | IL-10 expression as a relevant therapeutic. Schematic representation of GEMS receptor induction by RR120, resulting in IL-10 secretion.
After incubation and receptor induction, IL-10 concentrations were quantified using an IL-10 enzyme-linked immunosorbent assay (ELISA). Since the concentration of produced IL-10 was hard to estimate beforehand, and possible excessive IL-10 expression rate could not be measured, each sample was diluted several times. By measuring the dilutions, the IL-10 expression after receptor induction with RR120 could be quantified accurately (Figure 5).
Figure 5 | IL-10 expression as a relevant therapeutic. A HEK293T cells were transiently transfected with pLEO619, pLS13-IL-10, and pLS15, and subsequently activated with a single concentration of RR120 (300 ng/mL). Cells were incubated for minimally 40 h. Samples (100 µL) were taken directly from the cell medium, diluted, prepared according to the manufacturer’s instructions, and quantified using ELISA. To determine the IL-10 concentration both the samples’ absorbance (at 450 nm) and the calibration curve are consulted. Dashed bars represent IL-10 concentrations that were too high to be correctly quantified. Bars represent mean values of IL-10 concentrations (adjusted to individual dilution factors), overlayed individual data points represented as circles (for n=3 biologically independent samples). B Comparison between two values from Figure A.
The IL-10 expression for the 5x to 50x dilutions was too high to measure. For the 100x to 1000x dilution, an IL-10 concentration of approximately 9 pg/ml was seen (Figure 5A). For the 300x dilution of the sample that was treated with 300 ng/ml RR120, a 34-fold change in IL-10 expression was observed compared to the uninduced condition. Therefore, successful IL-10 expression was observed for all samples that were induced with 300 ng/ml RR120.
References