Our team, GEC-Guangzhou, has not only successfully constructed an efficient biosensing system but, more importantly, has established a complete, reusable technical framework and knowledge system. This provides comprehensive support for future iGEM teams, ranging from basic parts to practical applications. We systematically documented technical challenges, solutions, and management experiences encountered during the project advancement, forming a "plug-and-play" project template that will significantly lower the research threshold for future teams in the field of agricultural biotechnology. It is particularly noteworthy that our developed PQscR promoter, having undergone standardized characterization, can be directly integrated as a functional module into other teams' projects for rapid prototyping. Furthermore, we supplemented performance data and refined the dataset for the TnaA-FMO indigo colorimetric system, establishing its kinetic parameters and optimal working conditions under real-time detection settings. Future teams can directly reference this data to quickly construct visual biosensors.
Regarding biosafety, the "sample-in-answer-out" closed detection model we established provides a reusable biosafety paradigm for future teams. This design achieves physical containment of engineered bacteria through disposable detection tubes, coupled with strict waste disposal procedures, ensuring zero environmental release of genetically modified organisms. This safety strategy is particularly suitable for detection projects requiring field deployment. Future teams can directly adopt this architecture, adjusting the detection container specifications and incubation times according to their specific application scenarios.
Our systematic characterization of the PQscR promoter significantly expands its application scope. By validating its response characteristics to various AHL molecules, future teams can utilize it as a universal sensing module flexibly applied in different scenarios. For instance, it could be used to construct wound infection warning systems in the medical field [1], or to develop rapid detection tools for pathogenic bacteria in water for environmental monitoring [2]. The dose-response data and kinetic parameters we provide will help subsequent teams accurately predict system behavior and optimize circuit design. It is especially noteworthy that we have verified this promoter's capability to drive metabolic pathways, opening new avenues for building "sense-and-respond" systems.
This project provides systematic experimental data supplementation for the existing part FMO (BBa_K1131000) in the registry, offering crucial performance parameters and application guidance for future iGEM teams. Through rigorous time-course experiments and quantitative analysis, we established the complete kinetic profile of this colorimetric system under AHL induction: the optimal observation window is 6-8 hours, and the maximum yield under 1 µM AHL induction can reach 2.14 ± 0.14 mM.
Figure 1 Indigo Colorimetric Sensor. (A) Genetic circuit of the indigo colorimetric sensor. (B) Process of indigo production. (C) Indigo content after treatment with 1 µM 3OC12-HSL for different durations. (D) Indigo content after treatment with 1 µM C10-HSL for different durations. (E) Indigo content after treatment with 1 µM 3OHC10-HSL for different durations.
The "technology-product-application" translation model established by this project provides a complete exemplar for moving synthetic biology technologies from the laboratory to practical use. We not only developed a technical prototype but also designed detection procedures and supporting tools suitable for use by farmers. This user-oriented design approach can be referenced by future teams for other agricultural projects. Through in-depth communication with farmers, we have summarized key elements for transforming complex biotechnologies into simple operations, including visual result interpretation, simplified procedural steps, and design concepts adapted to field environments. These experiences hold significant reference value for promoting synthetic biology to address real-world problems.
[1]Kostakioti, Hadjifrangiskou, Hultgren.Bacterial biofilms: development, dispersal, and therapeutic strategies in the dawn of the postantibiotic era[J]. Cold Spring Harbor Perspectives in Medicine, 2013, 3: a010306.
[2]Ying Wu, Chien-Wei Wang, Dong Wang,et al.A Whole-Cell Biosensor for Point-of-Care Detection of Waterborne Bacterial Pathogens[J]. ACS Synthetic Biology, 2021, 10: 333−344.
