Parts
Overview

"To understand the whole it is necessary to understand the parts. To understand the parts, it is necessary to understand the whole. Such is the circle of understanding" – Ken Wilber

Introduction

All parts that have been used to develop !MPACT can be found on these pages. Here a brief summary can be found of all parts that have been used in our cell system. In the Basic Parts and Composite Parts pages, the parts that we have added to the registry are discussed in greater detail. Additionally, the Part Collection explains how each described part is used in our final design. The elaboration of the part GFP (BBa_I746909) that has been improved can be found on the Part Improvement page.


Part overview

Basic parts

Composite parts

Part collection

Part improvement

To develop the proof of concept of our cell therapy !MPACT, several Basic Parts have been designed and registered separately. Including several basic parts that were already in the Part Registry, we designed, tested, and registered multiple Composite Parts that were all codon-optimized for expression in HEK293T cells. All parts were synthetized by using IDT, as well as expression vectors offered by the Synthetic Biology research group of Eindhoven University of Technology. Furthermore, we created a Part Collection that gives a detailed overview of our complete proof of concept, containing the synthesized Generalized Extracellular Molecule Sensor (GEMS) receptors, followed by STAT3 expression and resulting in expression and secretion of either Secreted Alkaline Phosphatase (SEAP) or Interleukin 10 (IL-10). For Part Improvement, we designed a composite part to improve the basic part superfolder GFP (BBa_I746909). This part was codon-optimized for expression in E.coli. Our favorite parts are the basic part EpoR (BBa_K4160001) and the composite part GEMS receptor construct containing RR120 VHH as affinity domain (BBa_K4160008).


Type Part Number Name Description
Basic BBa_K4160000 Igκ secretion signal The Igκ secretion signal is a signal peptide that localizes the GEMS receptor to the cellular membrane.
Basic BBa_K4160001 EpoR EpoR is a type I cytokine receptor that initiates signal transduction when its ligand binds.1 The GEMS receptor is based on a mutated form of the EpoR.
Basic BBa_K4160002 IL6-RB IL-6RB is a trans-membrane protein that serves as the signal-transducing β-subunit of the interleukin 6 receptor.2 IL-6RB is fused to the EpoR of the GEMS receptor.
Basic BBa_K4160003 RR120 VHH RR120 VHH is the camelid heavy chain antibody A52 (VHHA52) fragment that is raised against the azo dye RR120.3
Basic BBa_K4160004 PR3 Proteinase 3 (PR3) is a neutrophilic serine protease that is involved in the pathogenesis of ANCA-associated vasculitis.4
Basic BBa_K4160005 STAT3 STAT3 is a transcription factor that binds to the STAT promoter to regulate STAT-induced gene expression.5
Basic BBa_K4160006 STAT promoter The STAT promoter that induces gene expression when binding to STAT3.5
Basic BBa_K4160007 IL-10 IL-10 is an anti-inflammatory cytokine.6 It is used as the therapeutic agent of our project.
Composite BBa_K4160008 GEMS receptor construct containing RR120 VHH as affinity domain This composite part combines BBa_K4160000, BBa_K4160001, BBa_K4160002, BBa_K4160003, and BBa_K2217015.
Composite BBa_K4160009 GEMS receptor construct containing PR3 as affinity domain This composite part combines BBa_K4160000, BBa_K4160001, BBa_K4160002, BBa_K4160004, and BBa_K2217015.
Composite BBa_K4160010 GEMS receptor construct containing PR3 as affinity domain with 8 amino acid linker This composite part combines BBa_K4160000, BBa_K4160001, BBa_K4160002, BBa_K4160004, and BBa_K2217015, containing a linker of 8 amino acids.
Composite BBa_K4160011 GEMS receptor construct containing PR3 as affinity domain with 31 amino acid linker This composite part combines BBa_K4160000, BBa_K4160001, BBa_K4160002, BBa_K4160004, and BBa_K2217015, containing a linker of 31 amino acids.
Composite BBa_K4160012 GEMS receptor construct containing PR3 fused to HA-tag as affinity domain This composite part combines BBa_K4160000, BBa_K4160001, BBa_K4160002, BBa_K4160004, BBa_K1150016, and BBa_K2217015.
Composite BBa_K4160013 GEMS receptor construct containing PR3 fused to HA-tag as affinity domain with 8 amino acid linker This composite part combines BBa_K4160000, BBa_K4160001, BBa_K4160002, BBa_K4160004, BBa_K1150016, and BBa_K2217015, containing a linker of 8 amino acids.
Composite BBa_K4160014 GEMS receptor construct containing PR3 fused to HA-tag as affinity domain with 31 amino acid linker This composite part combines BBa_K4160000, BBa_K4160001, BBa_K4160002, BBa_K4160004, BBa_K1150016, and BBa_K2217015, containing a linker of 31 amino acids.
Composite BBa_K4160015 GEMS receptor construct containing HA-tag as affinity domain This composite part combines BBa_K4160000, BBa_K4160001, BBa_K4160002, BBa_K1150016, and BBa_K2217015, containing a linker of 8 amino acids.
Composite BBa_K4160016 STAT-induced SEAP This composite part combines BBa_K4160006 and BBa_K1470004.
Composite BBa_K4160017 STAT-induced IL-10 This composite part combines BBa_K4160006 and BBa_K4160007.
Composite BBa_K4160018 UTR + sfGFP This part was developed for part improvement. The RBS in BBa_I746909 was replaced by BBa_K3972006 was added to..
Basic BBa_K1150016 HA-tag The HA-tag is a well characterized peptide that is used extensively in research as antibody epitope tag.7
Basic BBa_K2217015 bGH PolyA The bGH PolyA signal increases stability of RNA products by mediating efficient transcript termination and polyadenylation to heterologous genes.8
Basic BBa_K1470004 SEAP SEAP is a reporter protein that is useful to investigate the amount of transcriptional activity of enhancer/promoter elements.9
Basic BBa_I746909 sfGFP Folding of Superfolder GFP is enhanced relative to the folding of wild-tyoe GFP.
Basic BBa_K3972006 5’UTR with g10- RBS This part is used to improve the protein expression of part BBa_I746909. The part consists of translation-enhancing DNA, a poly-A-spacer, an RBS, and an AT-rich region.
Basic BBa_K3033009 eGFP eGFP is used as a control to check the transfection efficiency in the HEK293T cells.

Introduction

Igk secretion signal

EpoR and IL-6RB

RR120 VHH

PR3

STAT3 and STAT promoter

IL-10

References

To develop our proof of concept, seven basic parts have been designed and are explained in detail on the iGEM registry of parts. A brief description is found here.


Part Number Type Name Description Length Favorite
BBa_K4160000 Basic Igκ secretion signal The Igκ secretion signal is a signal peptide that localizes the GEMS receptor to the cellular membrane. 63bp
BBa_K4160001 Basic EpoR EpoR is a type I cytokine receptor that initiates signal transduction when its ligand binds.3 The GEMS receptor is based on a mutated form of the EpoR. 744bp

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BBa_K4160002 Basic IL-6RB IL-6RB is a trans-membrane protein that serves as the signal-transducing β-subunit of the interleukin 6 receptor.4 IL-6RB is fused to the EpoR of the GEMS receptor. 834bp
BBa_K4160003 Basic RR120 VHH RR120 VHH is the camelid heavy chain antibody A52 (VHHA52) fragment that is raised against the azo dye RR120.5 378bp
BBa_K4160004 Basic PR3 Proteinase 3 (PR3) is a neutrophilic serine protease that is involved in the pathogenesis of ANCA-associated vasculitis.6 663bp
BBa_K4160005 Basic STAT3 STAT3 is a transcription factor that binds to the STAT promoter to regulate STAT-induced gene expression.8 2321bp
BBa_K4160006 Basic STAT promoter The STAT promoter that induces gene expression when binding to STAT3.8 208bp
BBa_K4160007 Basic IL-10 IL-10 is an anti-inflammatory cytokine.10 It is used as the therapeutic agent of our project. 534bp

Igκ secretion signal (BBa_K4160000)

The Igκ secretion signal is a signal peptide that localizes proteins to the membrane of mammalian cells. Signal peptides are recognized when a secretory protein is translated by the ribosome. The signal peptide will bind the signal recognition particle, to form a complex that halts the translation and subsequently, is transported to the endoplasmic reticulum. From there, the translation will resume and the protein will eventually be secreted via the secretory pathway of the cell.1 In our project, BBa_K4160000 is used to translocate the synthetic receptor to the cell membrane.


EpoR (BBa_K4160001) and IL-6RB (BBa_K4160002)

The foundation of our project consists of the Generalized Extracellular Molecule Sensor (GEMS) receptor that was developed by Schneller et al., 2018.2 This receptor functions through dimerization of the extracellular receptor domains, leading to activation of the intracellular signal transduction domains.2 The extracellular receptor domain is based on a mutated form of the erythropoietin receptor (EpoR). EpoR is a type I cytokine receptor, that initiates signal transduction when its ligand, erythropoietin, binds. This induces reorientation and dimerization of two EpoR monomers to form a dimeric receptor structure (Figure 1).3


Figure 1 | Activation of the erythropoietin receptor. In the presence of erythropoietin, the EpoR activates through a conformational change between the two receptor subunits.

The mutation introduced to the EpoR renders it inert to erythropoietin.2 This mutated EpoR (BBa_K4160001) was fused to interleukin 6 receptor B (IL-6RB), which is an intracellular signal transduction domain. IL-6RB, also known as glycoprotein 130 (gp130), is a trans-membrane protein that serves as the signal-transducing β-subunit of the interleukin 6 receptor.4 Once the IL-6RB (BBa_K4160002) domains dimerize, downstream signaling of the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signaling pathway is induced.2,4


BBa_K4160001 is our favorite basic part because it forms the robust, modular GEMS receptor. To this EpoR, numerous affinity domains can be fused. Hence, this allows for the development of receptors for interaction with a wide range of targets. This way, our engineered cells can be altered to not only target autoantibodies specifically for vasculitis but also autoantibodies for other autoimmune diseases. Due to this modularity, the EpoR provides a platform that has much potential for therapeutics, which other iGEM teams can use effortlessly.


RR120 VHH (BBa_K4160003)

The basic part BBa_K4160003 encodes for RR120 VHH. This part is the camelid heavy chain antibody A52 (VHHA52) fragment that was raised against the azo dye RR120.5 In our project, the RR120 VHH domain is fused to the EpoR to activate the GEMS receptor in presence of the azo dye RR120 as a control for the general concept of receptor activation (Figure 2).


Figure 2 | Activation of GEMS receptor containing an RR120 VHH domain. In the presence of azo dye RR120, the GEMS receptor activates through a conformational change between the two receptor subunits.

PR3 (BBa_K4160004)

Proteinase 3 (PR3) is a neutrophilic serine protease that is present in neutrophil granules and at their membrane.6 PR3 degrades extracellular proteins at sites of inflammation and is known to be involved in regulating the immune system.7 Binding of the anti-neutrophil cytoplasmic antibody (ANCA) anti-PR3 to PR3 allows for activation of an autoimmune reaction that is responsible for ANCA-associated vasculitis.6 In our project, a truncated form of PR3 (BBa_K4160004) was fused to the EpoR to activate the GEMS receptor in the presence of anti-PR3 antibodies (Figure 3).


Figure 3 |Activation of GEMS receptor containing PR3. In the presence of anti-PR3, the GEMS receptor activates through a conformational change between the two receptor subunits.







STAT3 (BBa_K4160005) and STAT promoter (BBa_K4160006)

BBa_K4160005 encodes for the transcription factor Signal Transducer and Activator of Transcription 3 (STAT3) which is part of the STAT family. Phosphorylation of STAT leads to the dimerization of the monomers, which allows for binding to a specific DNA sequence. This binding event enables direct interaction with the STAT promoter (BBa_K4160006) that is located in front of STAT target genes, resulting in gene expression. STAT3 can be activated through activated receptor-associated kinase JAK.8 In our project, the expression of the target protein is regulated by STAT3 and its promoter. This promoter is accompanied by a cytomegalovirus (CMV) sequence, which is a commonly used enhancer element that allows for the high-level production of recombinant proteins in mammalian cells.9


IL-10 ((BBa_K4160007)

The part interleukin 10 (IL-10) (BBa_K4160007) is an anti-inflammatory cytokine. It has an essential role during infections as it inhibits the immune response during inflammation, to prevent damaging the host.10 IL-10 is recognized to have potential therapeutic effects on inflammatory diseases, including autoimmune diseases. 11 In our project, the IL-10 gene is controlled by the STAT promoter. Activation of the GEMS receptor will lead to IL-10 expression, resulting in its secretion by the engineered cells. IL-10 will behave as the therapeutic agent to inhibit the autoimmune reaction of ANCA-associated vasculitis. IL-10 is not on the White List of iGEM. However, we choose to implement IL-10 in our project, as multiple experts suggested the use of IL-10 as a very promising therapeutic strategy. We received permission from the Safety and Security Committee to implement IL-10 in our project.


  1. Güler-Gane G, Kidd S, Sridharan S, Vaughan TJ, Wilkinson TCI, Tigue NJ. Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids. PLoS One. 2016;11(5). doi:10.1371/JOURNAL.PONE.0155340
  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. Watowich SS. The Erythropoietin Receptor. J Investig Med. 2011;59(7):1067-1072. doi:10.2310/JIM.0B013E31820FB28C
  4. Wolf J, Rose-John S, Garbers C. Interleukin-6 and its receptors: A highly regulated and dynamic system. Cytokine. 2014;70(1):11-20. doi:10.1016/J.CYTO.2014.05.024
  5. Spinelli S, Tegoni M, Frenken L, Van Vliet C, Cambillau C. Lateral recognition of a dye hapten by a llama VHH domain. J Mol Biol. 2001;311(1):123-129. doi:10.1006/JMBI.2001.4856
  6. Granel J, Korkmaz B, Nouar D, et al. Pathogenicity of Proteinase 3-Anti-Neutrophil Cytoplasmic Antibody in Granulomatosis With Polyangiitis: Implications as Biomarker and Future Therapies. Front Immunol. 2021;12. doi:10.3389/FIMMU.2021.571933
  7. Sugawara S. Immune Functions of Proteinase 3. Crit Rev Immunol. 2005;25(5):343-359. doi:10.1615/CRITREVIMMUNOL.V25.I5.10
  8. Lee DS, Grandis JR, Johnson DE. STAT3 as a Major Contributor to Chemoresistance. Target Cell Surviv Pathways to Enhanc Response to Chemother. Published online 2019:145-167. doi:10.1016/B978-0-12-813753-6.00007-X
  9. Wang W, Jia YL, Li YC, et al. RETRACTED ARTICLE: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells. Sci Reports 2017 71. 2017;7(1):1-10. doi:10.1038/s41598-017-10966-y
  10. Saraiva M, O’Garra A. The regulation of IL-10 production by immune cells. Nat Rev Immunol. 2010;10(3):170-181. doi:10.1038/NRI2711
  11. Tian G, Li JL, Wang DG, Zhou D. Targeting IL-10 in Auto-immune Diseases. Cell Biochem Biophys. 2014;70(1):37-49. doi:10.1007/S12013-014-9903-X/TABLES/1

Introduction

RR120 VVH as affinity domain

PR3 as affinity domain

PR3 fused to HA-tag

HA tag

STAT induced

References

To develop our composite parts, we addressed the high modularity of the Generalized Extracellular Molecule Sensor (GEMS) system. The first composite parts that have been designed consist of several GEMS receptors. These receptors can bind to a specific target, resulting in the activation of downstream signaling. Different GEMS receptor constructs have been developed, which contain different affinity domains fused to the mutated form of erythropoietin receptor (EpoR) (BBa_K4160001), to bind different targets.


The composite parts that encode for the GEMS receptors contain a general structure (Figure 1) starting with an Igκ secretion signal (BBa_K4160000), followed by the modular affinity domain, EpoR (BBa_K4160001), IL-6RB (BBa_K4160002), and bGH poly A (BBa_K2217015), respectively. The Igκ signal peptide localizes the GEMS receptor to the membrane of mammalian cells. Subsequently, the affinity domain is followed by linkers of different lengths that fuse the affinity domain to the EpoR (BBa_K4160001). These linkers were incorporated to investigate whether the linkers significantly influenced receptor activation. The EpoR is the 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. When the affinity domain senses its ligand, the GEMS receptor undergoes a conformational change and consequently, dimerizes. As a result, the IL-6RB domain activates downstream signaling of the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway. At the C-terminus of all composite parts, a bovine growth hormone polyadenylation (bGH poly A) signal (BBa_K2217015) is located. bGH poly A mediates efficient transcript termination and polyadenylation to heterologous genes.1


Figure 1 | Overview of GEMS receptor design. The sequence encoding for this GEMS receptor is located in a pLeo619-Psv40 mammalian expression vector.

To express the GEMS receptors, a pLeo619-PSV40 mammalian expression vector is used (GenBank accession no. MG437012).2 With this expression vector, we have constructed GEMS receptors with different affinity domains. Further details about constructing the GEMS receptors with different affinity domains can be found in Experiments and in Notebook.


Part Number Type Name Description Length Favorite
BBa_K4160008 Composite GEMS receptor construct containing RR120 VHH as affinity domain This composite part combines BBa_K4160000, BBa_K4160001, BBa_K4160002, BBa_K4160003, and BBa_K2217015. 2322bp

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BBa_K4160009 Composite GEMS receptor construct containing PR3 as affinity domain This composite part combines BBa_K4160000, BBa_K4160001, BBa_K4160002, BBa_K4160004, and BBa_K2217015. 2600bp
BBa_K4160010 Composite GEMS receptor construct containing PR3 as affinity domain with 8 amino acid linker This composite part combines BBa_K4160000, BBa_K4160001, BBa_K4160002, BBa_K4160004, and BBa_K2217015, containing a linker of 8 amino acids. 2624bp
BBa_K4160011 Composite GEMS receptor construct containing PR3 as affinity domain with 31 amino acid linker This composite part combines BBa_K4160000, BBa_K4160001, BBa_K4160002, BBa_K4160004, and BBa_K2217015, containing a linker of 31 amino acids. 2675bp
BBa_K4160012 Composite GEMS receptor construct containing PR3 fused to HA-tag as affinity domain This composite part combines BBa_K4160000, BBa_K4160001, BBa_K4160002, BBa_K4160004, BBa_K1150016, and BBa_K2217015. 2627bp
BBa_K4160013 Composite GEMS receptor construct containing PR3 fused to HA-tag as affinity domain with 8 amino acid linker This composite part combines BBa_K4160000, BBa_K4160001, BBa_K4160002, BBa_K4160004, BBa_K1150016, and BBa_K2217015, containing a linker of 8 amino acids. 2651bp
BBa_K4160014 Composite GEMS receptor construct containing PR3 fused to HA-tag as affinity domain with 31 amino acid linker This composite part combines BBa_K4160000, BBa_K4160001, BBa_K4160002, BBa_K4160004, BBa_K1150016, and BBa_K2217015, containing a linker of 31 amino acids. 2702bp
BBa_K4160015 Composite GEMS receptor construct containing HA-tag as affinity domain This composite part combines BBa_K4160000, BBa_K4160001, BBa_K4160002, BBa_K1150016, and BBa_K2217015, containing a linker of 8 amino acids. 1988bp
BBa_K4160016 Composite STAT-induced SEAP This composite part combines BBa_K4160006 and BBa_K1470004 1771bp
BBa_K4160017 Composite STAT-induced IL-10 This composite part combines BBa_K4160006 and BBa_K4160007 772bp

GEMS receptor construct containing RR120 VVH as affinity domain (BBa_K4160008)

Our first composite part is the GEMS construct receptor fused to RR120 camelid heavy chain antibody VHHA52 (BBa_K4160003), which Scheller et al., 2018 developed to validate and optimize the GEMS receptor scaffold.3 In this composite part, RR120 VHH functions as the affinity domain. This domain is able to sense and bind its ligand azo dye RR120 (Figure 2). We implemented this composite part in our project to reproduce the GEMS receptor and analyze receptor transfection and activation.


Figure 2 | GEMS receptor constructs containing RR120 VHH as affinity domain. This receptor can sense the ligand azo dye RR120.

BBa_K4160008 is our favorite composite part as this GEMS receptor is readily reproducible. We successfully expressed this part in HEK293T cells and obtained significant activation after sensing its ligand RR120. In addition, the RR120 VHH domain can easily be replaced by other affinity domains, which signifies the modularity of the receptor and its ability to target a wide range of ligands. Therefore, this part proves the potential for therapeutics of the GEMS system.


GEMS receptor construct containing PR3 as affinity domain (BBa_K4160009, BBa_K4160010 & BBa_K4160011)

To investigate whether the GEMS receptor can be activated using an autoantibody as a ligand, we designed composite parts in which we replaced the RR120 VHH affinity domain with a truncated form of PR3 (BBa_K4160004). This affinity domain can sense and bind anti-PR3 antibodies. No papers have been published that describe GEMS receptor activation using antibodies as a ligand. Hence, we developed a library of composite parts that encode for the GEMS receptor containing the PR3 affinity domain fused to EpoR via different linkers. These parts include no (BBa_K4160009), an 8 amino acid (BBa_K4160010), and a 31 amino acid (BBa_K4160011) linker (Figure 3). These linkers mainly consist of repeats of glycine and serine, which is a classical linker motif for engineering multidomain proteins.4


Figure 3 | GEMS receptor constructs containing PR3 as affinity domain. A library of GEMS receptors containing a PR3 affinity domain was developed, including no, an 8 amino acid, or a 31 amino acid linker. These receptors should sense anti-PR3 antibodies.

GEMS receptor constructs containing PR3 fused to HA-tag as affinity domain (BBa_K4160012, BBa_K4160013 & BBa_K4160014)

The success of our designed library 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. We discuss this further on our Results page. As an alternative, we designed a library of composite parts that contain a hemagglutinin (HA) tag (BBa_K1150016) at the C-terminus of PR3. This HA-tag is a well-characterized peptide that is derived from the human influenza HA protein. It is used extensively in research as an antibody epitope tag, as it binds highly specifically to anti-HA antibodies.5 With this receptor construct, we could not only investigate whether the GEMS receptor could be activated by antibodies, in general, using anti-HA but also by disease-related antibodies using anti-PR3. Again, we developed a library of composite parts, which include no (BBa_K4160012), the 8 amino acid (BBa_K4160013), and the 31 amino acid (BBa_K4160014) linker (figure 4).


Figure 4 | GEMS receptor constructs containing PR3 fused to HA-tag as affinity domain. A library of GEMS receptors containing a PR3 with HA-tag affinity domain was developed, including no, an 8 amino acid, or a 31 amino acid linker. As illustrated, these receptors should sense the ligand anti-HA. In addition, they should also sense anti-PR3.

GEMS receptor construct containing HA-tag as affinity domain (BBa_K4160015)

In this composite part the GEMS receptor construct is fused to an HA-tag via an 8 amino acid linker (BBa_K4160015) (Figure 4). This part was designed as a control to investigate whether expression of the GEMS receptor on the cellular membrane was obtained, which is discussed further on our Results page. This part encodes for a DNA sequence that was used as a template for mRNA, which was produced and offered by our sponsor RiboPro. As the HA-tag could be produced immediately on the EpoR, there was no need to implement the PR3 at the N-terminus of this tag.


Figure 5 | GEMS receptors with HA affinity domain. This receptor should sense the ligand anti-HA.

Activation of the GEMS receptors induces activation of the JAK/STAT pathway. Therefore two composite parts were designed that encode STAT-induced proteins. These STAT-induced proteins are designed to function as the output of the GEMS system.




STAT-induced SEAP (BBa_K4160016) and STAT-induced IL-10 (BBa_K4160017)

For the first STAT-induced protein, we designed a composite part (BBa_K4160016) in which the STAT-promoter (BBa_K4160006) is fused to SEAP (BBa_K1470004). SEAP, an existing part we contributed to for the iGEM community, is a useful reporter protein to investigate the amount of transcriptional activity of enhancer/promoter elements.6 Therefore, this part was used to quantitively measure the activity of our GEMS receptors. To express this part, the mammalian reporter plasmid for STAT3-induced SEAP expression, pLS13, was used.7


For the second composite part (BBa_K4160017) we fused the STAT promoter to IL-10 (BBa_K4160007). This composite part was designed to achieve expression of the therapeutic agent IL-10. This part was also expressed using the expression vector pLS13 by replacing SEAP with IL-10.


  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. Expression vector pLeo619, complete sequence - Nucleotide - NCBI. Accessed September 8, 2022. https://www.ncbi.nlm.nih.gov/nuccore/MG437012
  3. 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
  4. Van Rosmalen M, Krom M, Merkx M. Tuning the Flexibility of Glycine-Serine Linkers to Allow Rational Design of Multidomain Proteins. Biochemistry. 2017;56(50):6565-6574. doi:10.1021/ACS.BIOCHEM.7B00902/ASSET/IMAGES/LARGE/BI-2017-00902M_0005.JPEG
  5. HA Tag Antibodies | Thermo Fisher Scientific - NL. Accessed October 3, 2022. https://www.thermofisher.com/nl/en/home/life-science/antibodies/primary-antibodies/epitope-tag-antibodies/ha-tag-antibodies.html
  6. Kain SR, Ganguly S. Overview of Genetic Reporter Systems. Curr Protoc Mol Biol. 2004;68(1). doi:10.1002/0471142727.MB0906S36
  7. Schukur L, Geering B, Charpin-El Hamri G, Fussenegger M. Implantable synthetic cytokine converter cells with AND-gate logic treat experimental psoriasis. Sci Transl Med. 2015;7(318). doi:10.1126/SCITRANSLMED.AAC4964/SUPPL_FILE/7-318RA201_SM.PDF
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