Parts: Expanding the Design Space of Protein Circuits

1. Coiled-Coil Domains: Introduced several coiled-coil (CC) domains into the iGEM Registry. These coding sequences serve as versatile motifs for mediating inter- or intramolecular protein-protein interactions, expanding the toolkit for synthetic protein circuit construction.
2. Trehalase Reporters: Submitted trehalase and split-trehalase sequences. Trehalase efficiently hydrolyzes trehalose into glucose, generating a quantifiable signal that supports glucose-based output designs for future biosensing applications.
3. Protease Modules: Added the protease AvrRpt2 and its specific cleavage site as a new regulatory element, enabling protease-dependent control in protein logic systems.
4. Composite Fusion Constructs: Developed fusion proteins combining CC domains with split reporters. Rearrangement or inhibition among CC modules regulates downstream domain reconstitution, achieving tunable activation or repression. Designed a multi-reporter system (luciferase, GFP, trehalase) for quantifying protein-protein interaction strength via luminescence, fluorescence, or glucose concentration.
5. Positive-Feedback Architecture: Constructed a feedback loop coupling CC-domain rearrangement with proteolytic cleavage. The reconstituted protease cleaves its own recognition site, enhancing protease concentration and reinforcing pathway output. The design functions as a modular scaffold for diverse protein-circuit applications.
6. Modular Scaffold for Protein Circuit Engineering: Established a unified protein-based framework that integrates CC-domain pairing, protease regulation, and reporter outputs. This system functions as a reusable scaffold adaptable to diverse protein circuit designs, facilitating modular logic construction and rapid biosensor prototyping.

For detailed information, please refer to our Parts page. We hope that our part collection can be explored and improved, contributing to the broader iGEM synthetic biology community.

Improvement on the Detection of Firefly Luciferase Luminescence

While using the Bac-Lumi™ Bacterial Firefly Luciferase Assay Kit (Beyotime), we observed a discrepancy between the manufacturer’s instructions and our results. The kit recommends a 5-minute incubation after reagent addition, but in our split-luciferase system, the luminescence signal decayed rapidly and returned to baseline within that time.

To address this, we performed continuous luminescence monitoring immediately after reagent addition, recording the signal every 30 seconds for at least 10 minutes. This allowed us to capture the true peak intensity.

Based on our observations, we recommend that future iGEM teams using luciferase—especially split-luciferase systems—perform time-resolved luminescence measurements within the first few minutes after reagent addition. This ensures accurate signal capture and prevents underestimation of luciferase activity.

Model

The contribution of our modeling work can be categorized into three aspects: epidemiological modeling, computational prediction, and molecular simulation.

At the epidemiological level, we extended the classical SEIR framework to describe the propagation dynamics of Pseudomonas syringae pv. tomato and incorporated detection-related parameters, developing the Crop-Spread (CS) model. This framework highlights the crucial role of early pathogen detection in disease control and can be generalized to other crop-pathogen systems with similar transmission mechanisms.

To support practical agricultural decision-making, we applied Q-learning, a reinforcement-learning approach, to identify economically optimal detection strategies. This enables farmers to balance cost and effectiveness in managing tomato diseases, enhancing both productivity and sustainability.

At the bioinformatics level, we built a specialized database of peptide-pair binding affinities, particularly focusing on coiled-coil domains—addressing a notable gap in existing peptide interaction data. This resource is open to the iGEM community and supports future protein-protein interaction studies.

We also developed Seq2Affinity, a QSAR-based regression and classification framework that predicts peptide-pair binding affinities directly from sequence information. By integrating ESM-2 embeddings with regularized linear models, it provides an efficient and interpretable pipeline for modeling sequence-function relationships in small-sample biological data.

At the molecular level, we used molecular simulations to validate our design:

1. CC-domain pairs adopt stable structural conformations, and differences in binding affinities are consistent with simulation results.
2. The AvrRpt2-RIN4 complex remains stable throughout the simulation, while its linker region retains the flexibility necessary for proteolysis.

These findings confirm the feasibility of our system and offer a standardized computational workflow—including PyMOL, Robetta, HADDOCK, GROMACS, and MMPBSA—for future iGEM teams to model biomolecular systems efficiently.

Hardware: Bringing Biosensing from Bench to Field

The Protato Kit is a modular hardware platform integrating sample grinding, collection, and biochemical detection into a single portable device. It bridges the gap between laboratory-based molecular sensing and field applications.

Traditionally, detecting Pseudomonas syringae in tomato plants requires specialized instruments and trained operators. In contrast, the Protato Kit measures potential changes at enzyme-modified electrodes, allowing low-cost, high-throughput, and on-site detection. Its 3D-printed housing provides both structural protection and a stable environment for biochemical reactions.

Key features include:

• Modular and extensible configuration - allowing additional modules as needed.
• Portable/integrated dual-version design - operates standalone or in an array for high-throughput detection; the integrated version supports temperature, humidity, and image monitoring for automated long-term analysis.
• User-friendly interface - enabling real-time visualization and data recording with simple operation.
• Low-cost production - suitable for stable replication and mass production.

For developers, we provide open-source CAD files (created in SOLIDWORKS) and circuit diagrams, enabling easy modification and reproduction using common 3D printers and components.

Beyond our project, the Protato Kit offers significant potential for other iGEM teams developing biosensors or agricultural monitoring systems, with flexibility to adapt to different enzymes, crops, or pathogens.

Software: A Local AI Framework for Smart Agriculture

We developed the Protato App, a user-friendly mobile platform that performs data acquisition, visualization, and AI-based analysis entirely on local devices, ensuring user privacy and offline functionality.

The app provides a modular framework for mobile-hardware integration and on-device computation, supporting flexible adaptation by future iGEM teams.

Main features:

• Cross-platform compatibility.
• Real-time connection with hardware devices via a local Wi-Fi network.
• Customizable data visualization and processing modules.
• On-device AI analysis for agricultural diagnostics.

We hope this framework can inspire future teams to build accessible, powerful, and privacy-preserving analytical tools for synthetic biology.

For more details, please visit our Software page.

Engagement: Science Popularization Booklet

As part of our Human Practices efforts, we created a popular science booklet titled "The Cell's Currency Gate — Transmembrane Transport", aimed at introducing complex biological concepts to the general public in an accessible and engaging way.

The booklet explains fundamental mechanisms of transmembrane transport, including simple diffusion, facilitated diffusion, and active transport, using vivid analogies such as “free channels,” “membership gates,” and “courier against the wind.” These metaphors help readers intuitively grasp how materials cross cell membranes, connecting molecular biology to familiar everyday experiences.

Our goal was to bridge the gap between professional knowledge and public understanding. We wrote and designed the content entirely by ourselves — from concept planning and text composition to illustration and layout — making it an original educational resource. Through this booklet, we hope to spark interest in cell biology and synthetic biology among young students and non-specialists.

We also made the booklet available in print and digital formats, allowing teachers and community educators to integrate it into classroom or outreach activities. By promoting biological literacy, we aim to inspire future iGEMers and cultivate a public that appreciates the role of synthetic biology in solving real-world problems.

→ The booklet can be downloaded from our Engagement page. Click to download

Looking Forward

Through our collective contributions in biological parts, computational modeling, hardware, and software, we aim to provide a foundation that helps future teams develop cell-free biosensors and field-deployable diagnostic systems.

We believe open, modular design is the key to making synthetic biology practical beyond the lab — from one tomato field to the next.