For the iGEM 2025 competition, our team focused on developing a synthetic biology toolkit to address the growing problem of pharmaceutical waste in Hong Kong. Our project was divided into two main goals: the detection and the removal of common medical pollutants. Through our work in the wet lab, we are proud to have made the following contributions to the iGEM community, including the submission of new BioBrick parts, the characterization of existing parts, and a functional proof-of-concept for our project goals.

1. Characterization and Comparison of Visual Reporters for Robust Biosensor Development

A key element of our detection system is a clear, visual output. To identify the most effective reporters for our E. coli chassis, we designed, constructed, and characterized expression systems for three different reporter proteins: mRFP1e (BBa_257EZ9BK), SYFP2 (BBa_2555QML9), and aeBlue (BBa_2538R3QI). We also created a co-expression strain to test the compatibility of mRFP1e + aeBlue (BBa_25FXVPFZ).

• Contribution: Our characterization studies provide valuable qualitative data for future iGEM teams. Through direct visual comparison of colony color and intensity, we confirmed that mRFP1e (BBa_K2796033) provides a significantly more robust and rapid visual signal in E. coli compared to other reporters like dTomato under our experimental conditions. This validates its engineered improvements for expression and maturation and provides a clear recommendation for teams seeking a strong, reliable red reporter protein.

2. A Novel Tetracycline Biosensor (BBa_25A81M7M) and New Characterization for an Existing Part (BBa_R0040)

As a proof-of-concept for our detection goal, we successfully designed and validated a whole-cell biosensor for the antibiotic tetracycline. In doing so, we also added valuable application data to a fundamental existing part from the Registry.

• Contribution:
• New Part: We have submitted our new composite part, BBa_25A81M7M, a tetracycline-inducible biosensor. Our functional studies demonstrate that this part produces a clear, concentration-dependent color change in response to tetracycline, establishing it as a functional tool for environmental monitoring.
• New Characterization for an Existing Part: Our biosensor was built using the well-established tetracycline-controlled promoter BBa_R0040. Our successful experiments serve as new application data for this part. We have added documentation to the BBa_R0040 part registry page, experimentally proving its effective use in a whole-cell biosensor system coupled with a chromoprotein output. This provides a validated, real-world use case for future teams looking to build upon this classic regulatory element.

3. A Novel Implementation Strategy: Immobilized Biosensors for Real-World Application

Recognizing the challenges of deploying engineered microorganisms, we proposed and validated a novel implementation strategy using alginate bead immobilization to improve the performance, safety, and reusability of whole-cell systems.

• Contribution: We successfully encapsulated our validated tetracycline biosensor cells in calcium-alginate beads. Our functional tests proved that the immobilized cells remained viable and responsive, producing a clear color change upon exposure to tetracycline. This work serves as a critical proof-of-concept, demonstrating that immobilization is a feasible strategy for creating contained, field-ready biosensing "cartridges." We contribute this methodology as a practical solution for future iGEM teams looking to bridge the gap between a lab prototype and a safe, real-world application.

4. Engineering an E. coli Strain for the Bioremediation of Tetracycline (BBa_25CS26NB)

To fulfill the "removal" aspect of our project, we engineered a synthetic E. coli strain capable of actively degrading tetracycline.

• Contribution: We have submitted a new basic part, BBa_25CS26NB, which expresses the TetX enzyme. Our contribution includes confirming its high-level expression via SDS-PAGE and demonstrating through functional assays that our engineered strain can effectively break down tetracycline in the external medium. This part provides the iGEM community with a validated tool for bioremediation.

5. Construction of a Foundational Two-Step Salicylate Degradation Pathway (BBa_257IE4RA)

As an extension of our bioremediation work, we aimed to engineer a pathway for degrading salicylate, a common metabolite of aspirin.

• Contribution: We successfully designed and constructed a synthetic operon for the co-expression of NahG and XylE. While we confirmed the co-expression of both proteins via SDS-PAGE, time constraints prevented full functional validation. However, we are contributing the fully assembled construct and our expression data. This provides a valuable and verified starting point for future iGEM teams to complete the characterization and implement this promising bioremediation pathway.

1. Optimized Alginate Bead Encapsulation Protocol

• Validated sodium alginate:water (1:60) and calcium chloride:water (1:10) ratios
• Proof-of-concept demonstration with lead-sensitive biosensor showing dose-dependent response
• Complete protocol for biosensor containment and signal enhancement

2. Open-Source fluidic Bead Formation System

• Two-module design (mixing + bead formation) with full design evolution documented
• Mixing module: S-shaped passive mixer achieving uniform viscous fluid blending
• Bead formation module: Flow-focusing design producing consistent spherical beads
• Complete CAD files, fabrication instructions, and testing protocols
• Low-cost fabrication using laser-cut acrylic sheets

3. Remedix: Integrated Portable Biosensing Laboratory

• Complete automated platform for culture, encapsulation, and detection
• Total development cost: ~USD $46.27
• Integrated components:
• Bacterial culture incubator with OD monitoring, temperature control, and UVC sterilization
• Automated fluidic bead formation
• AI-powered colorimetric detection using ESP32-CAM
• Real-time IoT alerts (SMS/WhatsApp)
• LCD touchscreen interface
• Detailed component list with cost breakdown

4. AI Vision System for Automated Color Analysis

• ESP32-CAM based image capture system
• Cloud-based AI processing pipeline (hosted on PythonAnywhere)
• Training methodology for chromoprotein color recognition
• Custom sieve assembly design to eliminate background interference

5. Complete Open-Source Code Repository

• MicroPython code for ESP32 boards (camera, motor control)
• Flask/Python backend for AI processing and live streaming
• C++ code for LCD touchscreen interface
• Figma app design for mobile monitoring
• All code available on GitLab: HongKong-JSS repository

1. Four-Stage Iterative Stakeholder Engagement Framework

• Stage 1: Problem Investigation – Mapping pharmaceutical residue sources and gaps
• Stage 2: Solution Design – Translating stakeholder needs into biosensor features
• Stage 3: System Integration – Expanding from device to complete platform
• Stage 4: Refinement & Future Directions – Identifying limitations and expansion pathways
• Clear documentation of how each stage shaped technical decisions

2. Comprehensive Stakeholder Engagement (30+ Consultations)

• 12 academic/scientific experts
• 6 industry practitioners (farmers, aquaculture)
• 3 government officials (DSD Director, AFCD, AMR Committee)
• 21 medical professionals (clinician survey)
• Public surveys and street interviews

3. Stakeholder-Driven Design Decision Map

• Documented table linking specific stakeholder feedback to technical responses
• Examples: Manual bead difficulty → millifluidic automation; Culture expertise barrier → integrated incubator

4. Quantified Public Awareness Data

• 83.3% discard expired drugs in trash (not pharmacies)
• Only 33.4% aware of local river pollution
• Low GMO understanding in environmental applications
• Baseline metrics for measuring education impact

5. Multi-Sector Application Framework

• Documented potential deployment pathways for Public, Industry, Science, and Government sectors
• Clear articulation of how Remedix serves different stakeholder needs

1. Extensive Educational Outreach (20+ Events, 4,000+ People)

• 2,500+ primary school students and parents
• 400+ secondary school students and teachers
• General public through media and community events

2. Complete Educational Toolkit (8 Resources)

• 3D-printed DNA puzzle model (with Tinkercad files)
• Proper drug disposal info cards
• Instagram educational campaign (some posts 2,000+ views)
• Scratch game: "DNA Duo"
• Blooket interactive quiz
• Educational worksheets
• Promotional stickers and posters
• All materials freely available

3. Documented Workshop Formats with Protocols

• Strawberry DNA extraction (110 participants)
• iGEM101: Molecular Biology intensive course (30 students, 3 days)
• Microscopy workshops ("Discovering the Invisible")
• Marine pollution LEGO STEM workshops
• Math x Science Challenge Cup (180 students, 8 schools)

4. Multi-Tiered Education Pipeline

• Elementary level: Basic microbiology and environmental awareness
• Junior high: DNA structure and biotechnology applications
• Senior high: Advanced lab techniques (restriction digestion, gel electrophoresis, transformation)
• Teacher training and inter-school collaboration events

5. iGEM Community Building Initiative

• Hong Kong High School iGEM Commemorative Ceremony (400 attendees)
• Cross-school collaboration platform
• Documented impact: 5 schools in iGEM101, new team interest from teachers

6. Media Outreach

• HK01 newspaper interview (general public)
• AM730 newspaper interview (STEM problem-solving)
• School broadcasts reaching 696 students