Along our iGEM journey, the team has set out to design a living therapeutic platform based on engineered probiotics for asthma treatment. Asthma is one of the most prevalent chronic respiratory diseases across the globe, with millions of patients taking daily medication that often does not manage symptoms, usually not addressing the root cause of the inflammation. Our project proposes a new strategy for biological delivery: using Lactobacillus plantarum, a safe probiotic harboring natural anti-inflammatory activity, engineered to deliver therapeutic RNA molecules directly to inflamed lung tissue.
Even from the beginning, our focus was not only on asthma but also on creating a modular platform that can be utilized for many pulmonary and gastrointestinal diseases. By sharing the design decisions along with any obstacles and solutions with the iGEM community, we hope to shed light on the path for future iGEM teams interested in synthetic probiotics and RNA therapeutics.
To bring this vision to life, we created multiple new basic and composite parts that can be used for various applications. All of them are listed on our [Parts Page]. Some of our most impactful parts are:
In our project, this part allows efficient Loading of therapeutic co-BERA into bacterial membrane vesicles and delivers it to Lung epithelial cells. We believe that this part represents not only a solution for our project but also a new building block for the iGEM community, a step toward programmable probiotics with precise molecular delivery capabilities.
The potential of this composite part goes far beyond our application:
Modular RNA loading system: DUF4811–L7Ae can be adapted to load different RNA scaffolds, making it useful for diverse RNA-based therapies.
Synthetic biology toolbox expansion: By linking RNA-binding proteins to transmembrane anchors, this design strategy provides a generalizable way to spatially localize RNA inside bacteria.
Therapeutic delivery:Future iGEM teams can reuse or modify this composite part to deliver RNAs, proteins, or other molecules via bacterial vesicles.
co-BERAs are highly stable RNA scaffolds that can simultaneously deliver multiple siRNAs. In our project, co-BERA was tailored to silence TSLP for asthma treatment, but the system itself is broadly applicable. Our team expanded the application of the co-BERA system by integrating it into a probiotic delivery platform.
co-BERA use goes beyond asthma: co-BERAs can be repurposed to target cancer drivers, viral infections, autoimmune pathways, or metabolic diseases. Future iGEM teams can adopt our composite design to load their own siRNA sequences into co-BERA, achieving precision. We provide the community with a versatile RNA-based therapeutic chassis that can serve as a general platform for synthetic biology applications.
In addition to designing new parts, we also worked on improving existing biological tools. One of the challenges in therapeutic delivery is ensuring that cargo such as RNA can escape the endosome after entering host cells. Natural Listeriolysin O (LLO) has this property, but its high virulence makes it unsuitable for therapeutic applications.
To overcome this, we engineered a mutated LLO variant with >100-fold reduced pathogenicity compared to the wild-type protein, while preserving its ability to mediate endosomal escape.
Why this matters: Endosomal escape is a major bottleneck in RNA therapy; only ~0.3% of internalized RNA typically escapes into the cytoplasm.
Our improvement: By modifying LLO, we provide a safer and more controllable tool for enabling RNA delivery without compromising host safety.
Impact: This improvement makes LLO compatible with probiotic-based therapeutics and provides a reusable part for the iGEM community interested in safe intracellular delivery systems.
We believe that this improved LLO part will be valuable beyond our project, enabling applications in RNA therapeutics, vaccine delivery, and engineered microbial therapies.
Safety is one of the fundamental considerations in designing our project. While working on the innovative therapeutic platform employing Lactobacillus plantarum, biosafety, to us, appeared equally important as just proving the functionality. Engineered microbes can do wonders for medicine and biotechnology, but they can be dangerous if released into any unintended environment or if they transfer their genes to other organisms.
To qualify the chassis to meet these requirements, our team incorporated multi-layer safety mechanisms, namely toxin-antitoxin kill switches and environmental sensors. These safety features serve to:
1. Prevent survival of engineered bacteria outside the intended site (lungs).
photo describtion
- PhoB Promoter(phosphate sensor): Ensures that bacteria cannot survive in the bloodstream, where phosphate levels are high.
- Thermosensor RNA: Detects temperature drop outside the host and kills the bacteria to avoid survival in the external environment.
2. Reduce the risk of horizontal gene transfer (HGT). One of the major biosafety concerns with GMOs (genetically modified organisms) is gene transfer to other microbes. By ensuring rapid elimination of engineered L. plantarum under non-permissive conditions, our system significantly reduces the window of opportunity for HGT.
Our multi-layered system generally offers a strategy that can be readapted by other iGEM teams. These tools take safety to a higher level without interference with the applications, whether for therapeutic probiotics, environmental biosensors, or biocontainment of industrial strains.
By editing and sharing these designs, we hope to contribute some practical biosafety tools to the iGEM community so that future teams can consider safety issues in their therapeutic and environmental projects.
Many iGEM teams use freeze-drying to prepare engineered probiotics as a stable powder for transport and storage. However, when bacteria are equipped with thermosensor-based kill switches, the sharp temperature decrease during freeze-drying can mistakenly trigger cell death. This creates a conflict: the very method needed to formulate the probiotic also eliminates the engineered strain.
To address this, we designed a freeze-drying preparation protocol that prevents accidental activation of the thermosensor-linked toxin–antitoxin system during manufacturing. This protocol allows engineered bacteria to survive freeze-drying while preserving the kill switch’s full function after rehydration in the host or exposure to non-permissive environments.
Problem-Specific Protocol– We provide the first documentation addressing the conflict between freeze-drying and thermosensor-based kill switches.
Generalizable Tool– Future iGEM teams developing safety circuits triggered by temperature can adapt our strategy to ensure their bacteria remain viable during preparation but safe in the environment.
Practical Resource – We share a protocol PDF that outlines design considerations and a workflow for integrating freeze-drying with thermosensor-equipped microbes.
How This Helps the Community
1. Teams can confidently apply freeze-drying without losing engineered strains prematurely.
2. Safety circuits remain reliable, preventing accidental survival outside intended conditions.
3. Our documentation saves time by providing a ready-to-use framework for others facing the same problem.
📄 Protocol PDF: [Insert Link to PDF File on Your Wiki]
As part of our project, we aimed not only to engineer a therapeutic system but also to create resources that benefit the broader iGEM community. One of our most impactful contributions is the development of a comprehensive molecular analysis and biosafety assessment platform that estimates the potential risk level of proteins before they are used in synthetic biology projects.
This advanced platform integrates multiple analytical capabilities to provide holistic molecular evaluation:
1. Cytotoxicity or pore-forming potential analysis (e.g., LLO variants)
2. Immunogenicity prediction (risk of triggering unwanted immune responses)
3. Horizontal gene transfer risk evaluation when combined with bacterial systems
4. Safety classification guidance with BSL level recommendations
5. Pathogenicity and genetic stability assessment
6. Environmental persistence and bioaccumulation analysis
1. 2D Structure Analysis: Detailed topology mapping, secondary structure prediction, and transmembrane region identification with confidence scoring
2. Pathway Mapping: Metabolic pathway associations, flux analysis, and drug interaction predictions with EC number classification
3. Domain Architecture Analysis: Protein domain identification, interdomain interactions, evolutionary conservation scoring, and ortholog analysis
4. Interaction Network Modeling: Protein-protein interaction prediction, regulatory network analysis, and network property calculations
5. Expert Review Workflow: Automated assignment of domain experts, review progress tracking, and regulatory compliance assessment
1. Multi-jurisdictional compliance checking (US, EU, International regulations)
2. CDC Select Agent screening and dual-use research evaluation
3. Export control assessment and institutional requirement analysis
4. Publication review recommendations and biosecurity risk evaluation
1. Multi-model risk prediction using Random Forest, XGBoost, Deep Neural Networks, and expert rule systems
2. Bayesian uncertainty quantification with confidence intervals
3. Real-time model performance monitoring and calibration metrics
4. Batch processing capabilities for high-throughput screening
1. Our platform comes with real-time 3D molecular visualization features that let us switch between rendering styles based on the analysis we need to do. SVG, PNG, and PDF are just a few of the high-quality formats in which we can export our visualizations when creating presentations or publications.
2. Our interactive analysis dashboards feature collapsible sections that we developed to help teams organize complex data without feeling overwhelmed. We also built in comprehensive metadata tracking and audit trails because we know how important it is to trace every step of our analysis process.
Preview to our advanced SynBio tool
By developing this platform, we hope to give iGEM teams in the future the regulatory guidelines, comprehensive molecular insights, and quantitative risk assessments we wish we had at the beginning of our project. We think that by taking a holistic approach, teams will be better able to choose proteins and plan experiments, which will ultimately result in safer experimental procedures.
We've seen too many teams spend valuable time in trial-and-error cycles with potentially harmful biological parts, so we wanted to create something that reduces those risks while promoting responsible innovation in synthetic biology research.
Our platform's modular architecture reflects our commitment to flexibility - we built it this way because we understand that different projects have different needs. The ensemble modeling approach we implemented ensures robust, evidence-based risk assessments, while our detailed confidence scoring and evidence tracking from multiple databases and literature sources maintains the transparency that we believe is crucial for building trust in computational tools.
We developed this platform not just as a technical solution, but as our contribution to making synthetic biology research safer and more accessible for teams who share our passion for responsible innovation.
Our Human Practices journey flowed along two parallel pathways, leading PRESS closer to society.
The first one was asthma awareness through songs that breathed compassion, games that taught through play, stories and posters that spoke of hope, guidebooks to navigate daily struggles, podcasts echoing real voices, and even a vision of an Asthma-Friendly City.
The other carried synthetic biology, its mysteries told through simple words, its power played in a card game, its promise unveiled in a guidebook, and its future glimpsed through educational reels.
Materials
Together, these streams merged, science with art, knowledge with imagination, reminding us that innovation is only complete when it belongs to the people it seeks to serve. None of that would have been possible without our outreach visits dedicating our efforts for society.
