At RDFZ-China, we believe that contribution means more than creating new parts — it means building upon existing knowledge, refining reusable tools, and sharing validated design principles with the entire iGEM community.
This year, our team’s contributions span genetic part improvement, safety validation, optogenetic toolkit construction, hardware development, Human Practices innovation, and team education resources.
We aimed to make our project not only a therapeutic prototype, but also a foundation upon which future teams can safely and efficiently engineer probiotic-based treatments.
I. Adding New Documentation to an Existing Part –
BBa_K1096002 (mazF)
1. Design Rationale
During the construction of our biosafety module, we noticed that many existing MazF toxin systems driven by Plac or IPTG-inducible promoters exhibit high basal leakage.
This leakiness often leads to premature toxin expression, causing host cell death and severely limiting strain propagation.
To overcome this, we redesigned the part by integrating MazF under the control of the arabinose-inducible promoter (PBAD).
The PBAD-mazF system provides:
- Tight repression in the absence of inducer (arabinose), ensuring safe cell propagation.
- Rapid and efficient cell death once induced, enabling strict containment.
- Standard compatibility with iGEM assembly (PBAD – mazF – B0015).
Figure 1: Schematic diagram of the PBAD-MazF construct and induction mechanism.
2. Experimental Construction and Verification
We synthesized a codon-optimized mazF gene and cloned it downstream of PBAD in the pSB1A3 vector, followed by the B0015 terminator.
The construct was transformed into E. coli DH5α via heat shock and verified by sequencing. Positive clones were screened on Ampicillin (100 µg/mL) LB plates.
To characterize the system, we cultured DH5α–PBAD–MazF under two conditions:
- Control group: No arabinose (verifying non-leaky safety).
- Induced group: 2% arabinose (triggering toxin expression).
Optical density (OD600) was measured at regular intervals to generate growth curves.
Figure : Growth curves showing tight repression in control group and growth inhibition in induced group.
3. Application and Value for Future Teams
The PBAD-MazF suicide system offers a standardized, tunable biocontainment device that future teams can easily reuse across multiple application fields:
- Therapeutic probiotics: Enables safe clearance of engineered bacteria from the human body post-treatment by administering arabinose as a non-toxic inducer.
- Environmental bioremediation: Provides an emergency shutdown mechanism for engineered strains released into open ecosystems.
- Industrial fermentation: Acts as a “life termination switch” to stop culture growth before product harvesting, reducing contamination risks.
By uploading new documentation, design rationale, experimental data, and safety analysis to the Registry entry of BBa_K1096002, we enriched the part’s reliability and usability for the entire iGEM community.
II. Hardware Contribution – Magnetically Controlled Red-Light Suppository
Our project integrates biological circuits with a hardware interface, forming a complete optogenetic therapy platform.
To achieve precise in vivo red-light induction, we designed a magnetically controlled red-light suppository, capable of safe and localized light delivery to the colorectal region.
Key contributions include:
- Innovative Design: A sealed, biocompatible capsule combining a red-light LED array and a Hall magnetic switch for wireless on/off control.
- Safety Optimization: Multi-layer epoxy encapsulation ensuring waterproof, pressure-resistant, and patient-safe operation.
- Interdisciplinary Framework: Combines synthetic biology control systems with hardware engineering, offering a template for future Bio-Hardware Interface designs.
Future teams focusing on optogenetics, biosensors, or therapeutic delivery can reuse our hardware structure as a flexible and validated light-induction platform.
III. New Genetic Toolkit for Future iGEM Teams
Beyond improving existing parts, we created a collection of new, modular parts for tumor-targeting, immune activation, and optogenetic control:
| Module |
Key Part |
Function |
| Targeting |
BBa_25PXJQ2S (INP-HlpA) |
Surface display system enabling EcN to adhere specifically to CRC cells via HSPG recognition. |
| Therapeutic |
BBa_25GADZ5E (YopE1-15-PD-L1) |
Secreted PD-L1 nanobody enabling local immune checkpoint blockade. |
| Coagulation |
BBa_256O7C4S (coa) |
Thrombin-like protein that blocks tumor blood flow. |
| Optogenetic Control |
BBa_25LPRH4Z (BphO) / BBa_25IOLYVT (PadC) / BBa_259UHA9L (NETMAP promoter) |
Red-light sensing and gene activation network. |
| Biosafety |
PBAD-MazF System |
High-tightness, inducible self-kill system ensuring safe use of engineered probiotics. |
Together, these parts form a reusable modular library that enables future teams to assemble controllable therapeutic circuits quickly.
IV. Human Practices and Educational Contributions
Throughout our project, we prioritized the responsible and educational development of synthetic biology:
- Public Engagement: Through activities like the Synthetic Biology Card Game (in collaboration with CAU-China), we developed accessible educational tools for younger students.
- Expert Consultation: We interviewed clinicians specializing in colorectal cancer, whose feedback guided both our project direction and ethical risk assessment.
- Outreach Resources: We documented our HP methodology and feedback pipeline as a reference for future high school teams.
These efforts collectively contribute to the broader iGEM community by demonstrating how to bridge synthetic biology research and social responsibility.
V. Collaboration and Community Impact
Our team actively collaborated with multiple domestic and international teams:
- With MPA-USA: Joint discussions on CRC therapeutic strategies and shared use of the mazF-based kill switch concept.
- With CAU-China: In-person visit and joint educational activity using their synthetic biology card set.
- With BNDS-China: Roundtable discussions on project management, modeling, and HP frameworks.
- At CCiC: Public presentation and poster exhibition, where we shared our design ideas and hardware innovations.
Through these collaborations, we promoted cross-border cooperation, biosafety awareness, and standardized communication for future iGEM teams.
VI. Team and Educational Resource Contribution
We also developed several internal tools and resources that will be shared with future iGEM high school teams:
- Modular DBTL Documentation Template: A structured form for recording design–build–test–learn cycles.
- Safety Training Manual: Tailored for high school-level synthetic biology labs.
- Hardware Assembly Guide: Detailed instructions for small-scale bio-hardware integration projects.
VII. Summary – Lasting Impact
Our contributions this year go beyond the scope of one project.
From improving a classic toxin system (BBa_K1096002), to creating new optogenetic and targeting modules, developing a novel hardware interface, and sharing validated HP and educational frameworks, we provide future iGEM teams with a complete, reproducible foundation for safe, light-controlled probiotic design.
Through our work, we hope to strengthen the iGEM community’s shared mission —
“Get, Give, and Share.”
