During our journey in the iGEM 2025 competition, our team committed to addressing the critical agricultural challenge of postharvest strawberry spoilage, focusing on the development of a VOC biosensor based on the PgrpE promoter and a complementary fungal control system. Through systematic experimental design and validation, we constructed and optimized highly sensitive VOC-responsive genetic elements and efficient fungal cell wall-degrading modules, accumulating robust experimental data and practical experience. These achievements not only enabled us to meet our project goals but also provide future iGEM teams with directly applicable synthetic biology tools and methodologies, aiming to reduce experimental exploration costs and accelerate project implementation. Our contributions are primarily reflected in three areas: bio-part registration and functional validation, experimental protocol optimization, and application strategy design. All results have been compiled into detailed documentation and uploaded to the iGEM Parts Registry.

We designed and validated a PgrpE promoter-based VOC biosensor. This part drives the violacein biosynthesis pathway in the presence of characteristic strawberry spoilage gases (e.g., 1-octen-3-ol), enabling visible detection of spoilage status. Concurrently, we constructed a fungal cell wall degradation system composed of chitinase (RmChi44) and glucanase (MoGluB). This dual-enzyme system effectively disrupts fungal cell structure through synergistic action, inhibiting the growth of common strawberry spoilage fungi. These parts underwent multiple rounds of optimization and functional validation, demonstrating high response efficiency and good host compatibility. They have been registered in the iGEM Parts Registry with comprehensive characterization data. We also optimized relevant experimental protocols, including enzyme activity assays and fungal inhibition test conditions, and compiled troubleshooting guides for common issues (e.g., low protein expression, insignificant inhibition zones) into a lab manual to assist future teams in getting started quickly and avoiding potential pitfalls.

Furthermore, we proposed an integrated strategy for strawberry spoilage monitoring and intervention that combines the biosensor and fungal control system, achieving closed-loop management from early spoilage detection to proactive fungal inhibition. The system is accompanied by detailed operating guidelines and application notes, facilitating the translation of laboratory results into practical scenarios. Our documentation, data, and guidelines provide reusable resources for the fields of food quality monitoring and sustainable agriculture. We hope this work will inspire future iGEM teams to further optimize system performance or expand its application to other postharvest disease control areas, collectively advancing synthetic biology towards solving real-world problems.

In this year's project focusing on the detection and control of postharvest strawberry spoilage, our team successfully designed, constructed, and systematically characterized various novel basic parts and composite parts, while also extending the functionality and improving the documentation of existing parts. These parts cover three core functional modules: VOC detection, visual reporter output, and fungal inhibition, forming a complete synthetic biology system from detection to intervention. Below is a brief overview of the primary parts:

• recA (BBa_K629001, Bronze): Served as a benchmark VOC-responsive promoter for evaluating the performance of other promoters. We significantly expanded its functional application and enhanced the iGEM Parts Registry records by adding new VOC response experimental data and detailed documentation.
• PgrpE (BBa_K338001, Gold): A highly sensitive VOC-sensing promoter capable of driving the violacein synthesis pathway, enabling visible detection of strawberry spoilage and providing an innovative solution for low-cost, equipment-free detection.
• RmChi44 (BBa_25SRZMPF, Silver): A chitinase derived from Rhizomucor miehei that specifically degrades chitin in fungal cell walls, offering a green and safe antifungal strategy.
• vioABCDE (Composite Part): The violacein biosynthetic gene cluster. Combined with PgrpE, it enables the visual conversion of VOC signals into a purple pigment.
• MoGluB (BBa_25QSVTMW): A glucanase that acts synergistically with RmChi44 to enhance fungal cell wall degradation.
• Other Parts: These include basic parts such as soxS (BBa_K554000), lasI (BBa_R0079), vioA, vioB, vioC, vioD, vioE, as well as the RBS (BBa_B0034) and terminator (BBa_B0015), which provided crucial support for system construction.

All these parts have been either newly registered or updated in the iGEM Parts Registry, accompanied by detailed part information, sequences, intended functions, and standardized characterization data. Through this year's work, we provide future iGEM teams working in areas like food safety, agricultural preservation, fungal disease control, and environmental monitoring with a comprehensive, reusable experimental resource library. These parts and methods will help other teams lower the barrier to entry, save time on preliminary exploration, and promote the implementation and adoption of synthetic biology in real-world production and applications.

Table 1 Component Registration Form

Bba	Part Name	Type	prize
BBa_K629001	recA（old）	basic-promoter	bronze
BBBa_K338001	grpE（old）	basic-promoter	gold
BBa_K554000	soxS（old）	basic-promoter
BBa_R0079	lasI（old）	basic-promoter
BBa_25SRZMPF	RmChi44	basic-coding	silver
BBa_25QSVTMW	Mo GluB	basic-coding
BBa_254RC0VZ	vioA	basic-coding
BBa_25QV4X3W	vioB	basic-coding
BBa_25DAPLUH	vioC	basic-coding
BBa_25294RVF	vioD	basic-coding
BBa_25DVIJ39	vioE	basic-coding
	vioA-B-C-D-E	composite
BBa_B0034	B0034	RBS
BBa_B0015	B0015	Terminator

Overview

When screening for novel VOC-responsive promoters, a benchmark promoter is essential as a control tool for evaluating the sensitivity and response range of target promoters. We conducted functional expansion research on the registered Bronze part BBa_K629001 (the recA promoter) to further explore its potential application in VOC detection scenarios. The recA promoter is a classic stress-responsive promoter from the Escherichia coli SOS repair system, typically activated under DNA damage or environmental stress. Its broad and predictable response characteristics make it an ideal control tool. In this project, we used recA as a benchmark to evaluate the VOC response performance of our core part, PgrpE. We have enhanced the content for BBa_K629001 in the iGEM Parts Registry by adding new experimental data and detailed documentation. These additions include comprehensive VOC response characterization data, experimental protocols, and application scenario descriptions, providing future researchers with more thorough reference materials and enriching the resource library of the iGEM community.

Basic Pathway Validation: recA-mRFP Verification

To further validate the functional characteristics of the recA promoter in VOC detection, we linked it to the red fluorescent protein mRFP, constructing a recA-mRFP reporter system and transforming it into E. coli BL21(DE3). We then exposed this engineered strain to three representative VOCs (1-octanol, 1-octen-3-ol, and phenylethyl alcohol) and measured its fluorescence output. The experimental results indicated that the activation of recA by VOCs was limited, with only a modest increase in the fluorescence/OD600 ratio, consistent with its expected performance as a control promoter. These data provide a reliable performance benchmark for comparing PgrpE and other promoters. Furthermore, we systematically compiled the experimental design, operational procedures, and characterization results, supplementing the BBa_K629001 page on the iGEM Parts Registry. This provides clear experimental guidance and data support for future teams intending to use this part.

Figure 1: Response of recA reporter strains to different VOCs

Overview

During the postharvest transportation and storage of strawberries, fungal infection is the primary cause of spoilage and economic losses. Volatile organic compounds (VOCs) produced by fungal metabolism are released during the early stages of spoilage, making them ideal early warning signals. To achieve real-time detection of these VOCs, we designed a VOC-sensing system centered around the PgrpE promoter.

The PgrpE promoter originates from the heat shock response pathway of Escherichia coli and is strongly activated under external environmental stress. We were the first to discover that PgrpE exhibits a significant response to VOCs released during strawberry spoilage and confirmed its sensitivity and application potential through a two-step validation strategy. Ultimately, we integrated it with a violacein-based colorimetric system to construct a visible early detection device for strawberry spoilage, providing a low-cost, rapid, and intuitive solution for fruit preservation.

Baseline Pathway Validation: PgrpE-mRFP Primary Verification

To verify whether the PgrpE promoter could effectively respond to VOC signals, we first constructed a reporter system (PgrpE-mRFP) with PgrpE driving the expression of the red fluorescent protein (mRFP).

Figure 2 Experimental Flowchart

In the experiment, the engineered strain was exposed to three major spoilage-related VOCs (1-octanol, 1-octen-3-ol, and phenylethyl alcohol). The results demonstrated that the mRFP expression driven by the PgrpE promoter was significantly higher than the control group under all VOC conditions. The most pronounced response was observed with 1-octanol and phenylethyl alcohol, showing a 2.3-fold increase in normalized fluorescence intensity. The response to phenylethyl alcohol was slightly lower but still substantial, while 1-octen-3-ol induced a comparatively lower, yet clearly elevated, response level.

Figure 3 Response of PgrpE-mRFP to different VOCs

Compared to other candidate promoters (soxS, lasI, recA), PgrpE demonstrated superior performance in both sensitivity and stability, confirming its feasibility as the core sensing element for VOC detection. This result established a solid foundation for subsequent development of a colorimetric visual output.

Full Pathway Construction: PgrpE-vioABCDE Final Validation

Following the primary verification confirming the high sensitivity of PgrpE, we coupled it with the vioABCDE violacein biosynthetic gene cluster. This integration enables VOC signals to be directly converted into a visible purple pigment, creating a visual detection system.

Figure 4 PgrpE responds to VOCs by producing violacein

In the experiment, we exposed the engineered strain to different concentrations of a VOC mixture (10 μL, 20 μL, 50 μL, 100 μL) and sampled at 0, 6, 12, 18, and 24 hours to measure violacein production. The results showed that violacein production exhibited a significant increasing trend with higher VOC concentrations and longer exposure times:

• At 10 μL, violacein accumulated slowly, reaching the lowest yield at 24 hours.
• At 50 μL and 100 μL, violacein production increased rapidly and was significantly higher than in the low-concentration groups after 12 hours.
• The peak production was reached at 24 hours, demonstrating a positive correlation between pigment yield and VOC dosage.

This result confirms that the PgrpE promoter can efficiently drive the vioABCDE gene cluster. Upon detecting strawberry spoilage-related VOCs, the system rapidly produces a distinct purple signal, enabling visual early warning of spoilage.

Through this two-stage validation—from fluorescent protein to colorimetric product—we successfully constructed a complete "detection-response-output" system, providing a novel synthetic biology solution for monitoring the fruit cold chain and managing spoilage.

Overview

Strawberries are frequently infected by fungi such as Botrytis cinerea and Rhizopus spp. during postharvest storage and transport. These pathogens rely on their robust cell walls to colonize and spread on strawberry surfaces. The fungal cell wall, primarily composed of chitin and β-glucan, provides high mechanical strength and environmental resistance.

While traditional chemical fungicides can inhibit fungal growth, they often pose risks of residue accumulation, drug resistance, and food safety concerns, making them unsuitable for widespread use on fresh fruits like strawberries. To address this challenge, we selected the chitinase RmChi44, sourced from Rhizomucor miehei. This enzyme specifically hydrolyzes the β-1,4-glycosidic bonds in chitin within the fungal cell wall, molecularly compromising the structural integrity of fungi, thereby reducing their infectivity and offering a green and safe control strategy.

Figure 5 Antifungal Mechanism of Chitinase

Baseline Pathway Validation: RmChi44 Primary Verification

First, we constructed the RmChi44 gene into an IPTG-inducible expression vector using cloning technology and transformed it into E. coli BL21(DE3). Following IPTG-induced expression, we detected its expression in the cell lysate.

In SDS-PAGE analysis, we observed a distinct protein band at the expected position, with a molecular weight consistent with the theoretical value for RmChi44. This confirmed the successful expression of RmChi44 in E. coli. This experiment completed the basic expression verification of RmChi44 in the engineered strain, laying the foundation for subsequent purification and functional testing.

Figure 6 Verification of RmChi44 engineered bacteria and SDS-PAGE validation

Full Pathway Construction: Functional Validation of Fungal Inhibition

Following confirmation of successful RmChi44 expression, we proceeded with enzyme purification and functional validation.

The purified chitinase demonstrated significant degradation of chitin structures when incubated with fungal cell wall substrates in vitro, resulting in clear damage to the fungal cell wall integrity. These results confirm that RmChi44 is not only successfully expressed but also maintains its biological activity, exhibiting potent inhibitory effects against Botrytis cinerea and soft rot pathogens (Rhizopus spp.).

Consequently, we propose an application strategy integrating RmChi44 into strawberry postharvest management:

The purified enzyme preparation can be sprayed onto strawberry surfaces. This approach enables precise targeting and disruption of fungal cell walls without damaging the strawberry tissue itself, thereby effectively delaying the spoilage process. This strategy presents a green alternative to conventional chemical fungicides and demonstrates significant potential for broader application.

Figure 7 Functional verification of chitinase and glucanase expression

In this year's project, our team designed and constructed several key parts for the first time and successfully validated their functions within an integrated system for detecting and controlling postharvest strawberry spoilage. We constructed the PgrpE promoter part (BBa_K338001) and verified its high sensitivity to VOCs released during strawberry spoilage using a red fluorescent protein (mRFP) reporter system (PgrpE-mRFP). Furthermore, we combined PgrpE with the violacein biosynthesis pathway (vioABCDE) to achieve the conversion of VOC signals into a visible purple pigment (BBa_25FGPJG1), providing an effective tool for equipment-free, low-cost visual detection of spoilage.

Additionally, we successfully constructed and validated the chitinase part RmChi44 (BBa_25SRZMPF), which specifically degrades the chitin structure in fungal cell walls, enabling a green and safe strategy for fungal control. We also utilized the recA promoter part (BBa_K629001) as a control element to standardize the performance evaluation of different VOC-responsive promoters.

Overall, our work establishes a comprehensive biological solution spanning from spoilage signal detection to control intervention. We provide detailed DNA sequences, experimental data, and standardized operating procedures. These resources will offer substantial support and reference for future teams working in areas such as food safety, cold chain management, and fungal disease control.

Frequently Asked Questions and Troubleshooting

During our experiments, we identified several steps prone to errors and propose corresponding solutions to assist other teams in avoiding common pitfalls:

Table 2 Common Problems and Solutions

Problem	Possible Cause	Suggested Solution
VOCs detection results fluctuate significantly	VOCs volatilization or unstable concentration	Operate in a fume hood and prepare standard stock solution in advance
Violet coloration is not obvious	RBS or promoter strength is insufficient	Replace with a stronger RBS or construct a dual-promoter system
Low protein expression	IPTG concentration is inappropriate	Optimize IPTG concentration and induction temperature in gradients

• Cross-Species Application: The PgrpE promoter can be widely applied in spoilage or environmental monitoring projects for other crops, such as tomatoes, citrus fruits, or cereals during transport.
• Food Safety Monitoring: Integrating the vioABCDE module with the PgrpE promoter enables visible spoilage detection suitable for scenarios lacking professional equipment.
• Green Agricultural Control: The RmChi44 and MoGluB parts can be utilized in other fungal-related projects, such as controlling rice sheath blight or grain mold.

1. Poria, V.; Rana, A.; Kumari, A.; Grewal, J.; Pranaw, K.; Singh, S. Current Perspectives on Chitinolytic Enzymes and Their Agro-Industrial Applications. Biology 2021, 10, 1319. https://doi.org/10.3390/biology10121319
2. Ngolong Ngea GLN, Qian X, Yang Q, et al. Securing fruit production: Opportunities from the elucidation of the molecular mechanisms of postharvest fungal infections. Compr Rev Food Sci Food Saf. 2021; 20: 2508–2533. https://doi.org/10.1111/1541-4337.12729
3. Yang, S., Fu, X., Yan, Q., Jiang, Z., & Wang, J. (2016). Biochemical Characterization of a Novel Acidic Exochitinase from Rhizomucor miehei with Antifungal Activity. Journal of agricultural and food chemistry, 64(2), 461–469. https://doi.org/10.1021/acs.jafc.5b05127