CONTRIBUTION

In our project, we designed and characterized a series of molecular parts that together construct the foundation for enhancing nitrogen fixation and nematode resistance in leguminous plants. These parts extend the iGEM Registry with new biosynthetic enzymes, regulatory elements, and toxin modules applicable to sustainable agriculture and plant–microbe interaction studies.

1. Luteolin Biosynthesis Pathway

   To reconstruct the luteolin biosynthetic pathway, we combined enzymes from Rhizobium leguminosarum, Escherichia coli, and Bacillus subtilis into modular plasmids. The following parts were designed, documented, and submitted to the iGEM Registry:

   Table 1 Luteolin Biosynthesis Pathway

   Part Name	Link	Description
   Rhizobium leguminosarum malonyl CoA synthetase (matB)	BBa_25oscpns	Facilitates activation of malonate, supplying precursors for flavonoid synthesis.
   Rhizobium leguminosarum malonate carrier protein (matC)	BBa_25vp9bp7	Transports malonate into the cytoplasm to support CHS/CHI reactions.
   Chalcone Synthase (CHS)	BBa_256kcp4y	Catalyzes condensation of 4-coumaroyl-CoA and malonyl-CoA into naringenin chalcone.
   Chalcone Isomerase (CHI)	BBa_25afp585	Converts naringenin chalcone into naringenin, a central intermediate in flavonoid biosynthesis.
   Tyrosine Ammonia Lyase (TAL)	BBa_25cmfdqn	Converts L-tyrosine into p-coumaric acid, linking amino acid metabolism to flavonoid biosynthesis.
   4-coumarate:CoA ligase (4CL)	BBa_251iopis	Activates p-coumaric acid to form 4-coumaroyl-CoA, a substrate for CHS.
   Flavone Synthase (FNS)	BBa_25q8x69f	Catalyzes the conversion of naringenin into apigenin and subsequently luteolin.
   Flavonoid 3′-Hydroxylase (F3′H)	BBa_25lr6ufr	Hydroxylates apigenin to luteolin, completing the flavonoid pathway.
   LoxP-Terminator (T7 terminator and rrnB T1 terminator) - loxP	BBa_25rhpd9d	Composite regulatory element integrated into the Cre-loxP system for controlled gene expression.

   
   
   
   
2. Nematode-Resistant Modules

   To build nematode-resistant systems, we engineered Bacillus subtilis to express Cry region proteins and their fusion derivatives, optimizing them for safe and controllable environmental application.

   Table 2: Nematode-Resistant Modules

   Part Name	Link	Description
   Cry6Aa protein	BBa_2518kpzo	A pore-forming toxin lethal to nematodes via gut cell lysis.
   Cry5Ba protein	BBa_25bm27wd	Targets nematode intestinal cells to disrupt epithelial integrity.
   Cry6Aa–Cry5Ba Fusion Protein Gene	BBa_25niecs5	A novel chimeric construct combining two Cry proteins for enhanced nematode toxicity.

   
   
   
   
3. Community Impact

   All designed parts have been documented following iGEM standards, including sequence verification and experimental validation. They collectively expand the iGEM Registry with agricultural synthetic biology modules focused on nitrogen fixation and biotic stress resistance, offering new tools for flavonoid pathway engineering and biocontrol research. We hope these open-source parts will support future teams in developing sustainable solutions for crop yield improvement and soil health management.

Our hardware design represents a practical and innovative contribution to the iGEM community by addressing one of the most persistent challenges in synthetic biology applications: the lack of field-operable detection systems capable of providing real-time feedback for engineered biological circuits. During the development of our project, we recognised that the regulation of salicylic acid concentration played a decisive role in controlling the secretion of fusion proteins by our engineered bacteria. However, existing methods for measuring salicylic acid relied heavily on laboratory-based chemical assays that were time-consuming, required specialized equipment, and failed to meet the demands of rapid on-site analysis. To bridge this gap, we designed and constructed a modular, low-cost, and portable detection tube that enables rapid and semi-quantitative measurement of salicylic acid in soil. By translating a complex analytical process into a simple field-deployable system, our hardware allows synthetic biology to move beyond the laboratory and into real-world environments.

The detection apparatus integrates an external protective sleeve and an internal test tube that houses the salicylic acid-specific detection strip. When inserted into the soil and flushed with water through the top injection inlet, the sleeve facilitates the formation of a soil leachate, which then contacts the detection strip inside the test tube. The resulting color change allows users to visually estimate the concentration of salicylic acid on site, providing immediate feedback about the soil’s biochemical status and the operational state of the engineered microbes. The system’s mechanical structure was refined through multiple design iterations to improve its functionality, durability, and ergonomics. After expert consultation and prototype testing, the sleeve diameter was reduced to 20 mm and the length extended to 250 mm, a design that both minimized resistance during insertion and ensured detection coverage within the typical root zone of leguminous plants. The introduction of a countersunk hole structure within the sleeve further enhanced stability and protected the test tube from mechanical stress during use.

Our design process also emphasized the principles of modularity and maintainability. The test tube was divided into four detachable components connected through a snap-fit locking mechanism, allowing easy replacement of detection paper and filtration layers. This modular concept not only improved the maintainability of the hardware but also provided a flexible foundation for future adaptation. The structure was optimized through a series of improvements—from revising the initial oversized prototypes to developing a more streamlined form compatible with field operations and 3D-printing constraints. Each modification, including the adoption of a top-injection design instead of a side-pipe structure, was guided by practical testing and feedback from hardware experts and professors. These iterative refinements ensured that the final version achieved a balance between mechanical robustness, functional precision, and user-friendly assembly.

The contribution of this hardware to the iGEM community lies in its reusability, adaptability, and potential to serve as a foundational model for future biosensing devices. All CAD models, assembly blueprints, and material parameters will be made openly available, allowing future teams to replicate, improve, or repurpose the system for different detection targets. By replacing the salicylic acid test paper with other functional strips or biosensor modules, the device can be applied to detect diverse analytes such as plant hormones, soil nutrients, or heavy metals. Its design can also be easily modified to accommodate different root-zone depths or soil types, making it a versatile platform for environmental monitoring and agricultural biotechnology. This open, modular, and low-cost approach exemplifies the collaborative spirit of the iGEM community, empowering future teams to integrate hardware and biology for real-time feedback in synthetic ecosystems.

Ultimately, our hardware represents more than a supporting tool—it demonstrates how engineering design can strengthen the interface between synthetic biology and the environment. By contributing a robust and field-ready salicylic acid detection system, we provide the community with a concrete example of how mechanical design, material selection, and biological function can be harmonized into a unified experimental platform. We hope that this device will inspire other teams to pursue similarly integrative designs that enable synthetic biology to operate safely and effectively in natural contexts, promoting a future where engineered biological systems can dynamically interact with and respond to their environments.