Part
Collection
"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
Therapeutic expression
Antibody-induced activation
Overview parts
References
Introduction
!MPACT is based on the Generalized Extracellular Molecule Sensor (GEMS) system which is developed by Scheller et al., 2018.1 This is a robust and modular platform that allows mammalian cells to sense a wide range of extracellular molecules, which induce a customizable response.1 This Part Collection contains all of the parts that have been used to develop the GEMS platform of !MPACT. More information about !MPACT can be found on the Project Description page.
In our project, we demonstrate two applications of the GEMS platform: expression of a therapeutically relevant protein, and antibody-induced GEMS receptor activation. These applications are discussed in detail on our Proof of Concept page. Our Part Collection consists of the core modules to design the tailored synthetic GEMS receptor constructs. This collection clearly establishes the modularity of the GEMS platform, confirming a remarkable tool that has much potential for therapeutics. We believe that this will greatly contribute to the iGEM community.
Therapeutic expression
Different GEMS systems were developed to test the design of !MPACT. The therapeutic expression system achieves interleukin 10 (IL-10) secretion after induction of the GEMS system with its ligand RR120. This system was designed by combining the GEMS receptor construct containing RR120 VHH as affinity domain (BBa_K4160008), Signal Transducer and Activator of Transcription 3 (STAT3) (BBa_K4160005), and STAT-induced IL-10 (BBa_K4160017). When sensing and binding RR120, the GEMS receptor construct undergoes a conformational change and consequently, dimerizes. This activates the Janus Kinase (JAK)/STAT pathway. Subsequently, this induces the expression of STAT3 which, thereafter, promotes STAT-induced IL-10 expression and secretion (Figure 1). Visit our Results page to see the promising results for our therapeutic expression system.
Antibody-induced GEMS receptor activation
To achieve activation of the GEMS system using antibodies, two GEMS systems have been developed. In the first system, the affinity domain consists of PR3, without or with an amino acid linker (BBa_K4160009, BBa_K4160010 & BBa_K4160011). This receptor construct is combined with STAT3 and STAT-induced SEAP (BBa_K4160016) Sensing and binding the anti-PR3 antibodies results in STAT-induced SEAP expression and secretion via the JAK/STAT pathway (Figure 2).
The second system encompasses GEMS receptor constructs containing an additional HA-tag fused to the PR3 affinity domain. (BBa_K4160012, BBa_K4160013 & BBa_K4160014). Also for this system, the receptor construct is combined with STAT3 and STAT-induced SEAP. In addition to anti-PR3 antibodies, these receptors can bind anti-HA antibodies (Figure 3).
Antibody-induced activation of the GEMS receptor constructs remains challenging but is actively under investigation. Our initial results can be found on the Results page.
Overview
Altogether, Figure 4 shows an overview of all parts that were used to develop !MPACT. These parts clearly demonstrate the modularity of the GEMS platform.
| Part Number | Type | Name | Description | Length |
|---|---|---|---|---|
| BBa_K4160005 | Basic | STAT3 | STAT3 is a transcription factor that binds to the STAT promoter to regulate STAT-induced gene expression.2 | 2321bp |
| 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 |
| 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 8 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_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 |
- 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
- 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