Wetlab
Establishing a Binder-Based Diagnostic Platform
To achieve early diagnosis of rhinosinusitis, our team developed a colloidal gold–based rapid test strip designed to detect the key biomarker Granzyme K (GZMK). This design not only demonstrates the feasibility of de novo–designed proteins in diagnostic applications but also provides the iGEM community with a replicable, scalable, and low-cost diagnostic framework.
First, we proposed a modular “binder-instead-of-antibody” strategy. Traditional colloidal gold assays rely on specific antibodies, whose production is costly, time-consuming, and dependent on animal experiments—posing limitations in educational or resource-constrained research environments. To overcome these challenges, we employed computational protein design technologies to create novel protein binders that specifically recognize GZMK, successfully replacing antibodies in the test strip. This approach significantly lowers the barrier to diagnostic development and provides a generalizable path for specific detection without antibody dependence.
Second, we established an innovative “Binder1–GZMK–Binder2” detection architecture. Compared to the conventional “target–antibody–antibody” model, this structure aligns more naturally with de novo design principles. By simply replacing the target sequence and redesigning the binders, the system can be easily adapted to detect various biomarkers, making the entire platform highly flexible and extensible.
Through these innovations, we achieved rapid and specific detection of GZMK and, more importantly, contributed a reusable “Binder-based Test Strip” construction framework to the iGEM community. We systematically optimized parameters such as coupling pH, binder-to-gold ratio, and membrane coating conditions, and we will share standardized operating protocols on our Measurement page. This contribution provides future teams with a practical reference for implementing binder-based diagnostic tools, promoting the broader application of synthetic biology in diagnostic technology development.
Developing a Standardized GZMK Activity Assay
To verify whether the expressed and purified GZMK possesses the expected enzymatic activity, and to provide a stable and reproducible foundation for subsequent inhibitor screening, we established a high-sensitivity in vitro enzymatic activity assay system. This system not only confirms the functional activity of the protein but also enables quantitative evaluation of its catalytic characteristics through kinetic parameters, thereby supporting high-throughput screening and mechanistic studies of potential inhibitors.
After comparing multiple detection approaches, we selected a fluorogenic substrate (DABCYL–GDGRSIMTE–EDANS) that provides superior specificity and signal-to-noise ratio, and constructed an assay based on the principle of FRET (Fluorescence Resonance Energy Transfer). By systematically optimizing buffer composition, enzyme and substrate concentrations, and reaction conditions, we successfully obtained a strong and reproducible enzymatic signal from recombinant GZMK, confirming its catalytic functionality. Furthermore, by performing substrate concentration gradient experiments, we plotted the enzyme velocity–substrate concentration curve and derived key kinetic parameters (Km and Vmax), thereby establishing a quantifiable, robust, and high-throughput-compatible standardized assay platform.
The design and optimization process of this system provides a valuable reference framework for future iGEM teams, and we will share standardized operating protocols on our Measurement page. Other teams can readily adapt this approach by modifying substrate sequences or reaction conditions according to their target enzymes, allowing rapid establishment of customized activity assays. This contribution not only expands the application of synthetic biology in enzymology and drug screening but also serves as a model for experimental standardization and community sharing.
Innovating a Safe Expression Strategy for Cytotoxic Proteases
GZMK is a key pathogenic factor and potential diagnostic biomarker in chronic rhinosinusitis. However, its toxicity both inside and outside the cell, along with its dependence on proper structural folding for enzymatic activation, makes stable expression in conventional systems extremely challenging.
Building upon previous studies, we established and optimized a novel strategy for GZMK expression and purification. In this approach, GZMK is secreted as an inactive zymogen from HEK 293F cells, allowing safe accumulation in the culture medium. The precursor protein is then purified and activated in vitro by specific proteolytic cleavage to remove the N-terminal propeptide, yielding fully active mature GZMK with complete catalytic functionality.
This method not only enables efficient and stable expression of GZMK in a eukaryotic system but also provides a generalizable and safe expression framework for potentially cytotoxic proteases. Future teams can adapt this strategy by modifying the signal peptide, activation mechanism, or host system to facilitate the study or production of other active enzymes and functional proteins.
Detailed construct design and sequence information can be found in the Parts Registry, while the full expression and purification workflow is available on the Experiments page.
Contributing Multiple Functional Binder Parts
Our colloidal gold test strip utilizes de novo–designed protein binders to achieve specific recognition of GZMK. Through computational protein design and multiple rounds of screening, we successfully obtained several binders with high affinity toward GZMK. These binders serve as the key recognition modules in our detection system, effectively replacing traditional antibodies in the colloidal gold assay.
Beyond their application in this project, these binders provide reusable resources and methodological references for future teams working on protein binder design, protein–protein interaction studies, and functional analysis of GZMK or related enzymes. Each binder has been systematically expressed, purified, and validated following standardized protocols, and the corresponding functional parts have been registered in the Parts Registry for easy access and further extension by other teams.