Physical Injuries: We handle burns with 15-minute cold water rinses and burn cream, cuts with immediate cleaning and bandaging, and chemical exposure with water rinses and contaminated clothing removal. Our team practices fire protocols regularly.
Personal Protection: All members of our team must wear long lab coats, pants, gloves, masks, and tied hair without jewelry. We wash our hands before and after lab work, inspect equipment before use, supervise studies, and record everything our team performs.
Escape Prevention: We developed an olive compound system (150-300 mg/L hydroxytyrosol, 200-400 mg/L oleuropein) that kills escaped bacteria within minutes and biodegrades in 14-21 days. Our temperature sensor kill the bacteria at the environmental temperature, below 25°C, as our RNAT structures were engineered to make conformational changes at the low temperature.
Gene Transfer Prevention: We built a PemK-PemI addiction system that makes our bacteria dependent on our plasmid, they die if they lose it because the protective protein, which is our antitoxin (PemI), degrades faster than the endoribonuclease, which is our toxin (PemK). This ensures our engineered bacteria can't survive without our control systems.
Growth Control: We incorporated built-in quorum sensing that only activates at 10⁹ CFU/ml, keeping our bacteria quiet at lower densities and preventing uncontrolled multiplication.
Targeting Accuracy: We designed computer-guided siRNA specificity using siDirect 2.0, our selective packaging through L7Ae-C/D Box systems, and our siRNA breaks down safely after its target. Our team ensures only intended target (TSLP-mRNA) are affected.
Controlled Release: We created pH-responsive activation only at acidic asthma conditions (pH < 6.9) and enzyme blockers preventing premature particle breakdown. Our system waits for the right conditions before activating.
Reduced Toxicity: We modified our LLO-L461T protein to be 100x less toxic than original, only activating when our sensors detect both low pH and high inflammation markers in the lungs where we need treatment.
Bloodstream Prevention: Our phosphate sensors kill bacteria in high-phosphate blood (0.8-1.4 mM) while allowing survival in low-phosphate lungs (0.2-0.4 mM). We also built temperature sensors that eliminate our bacteria outside body temperature, ensuring they can't survive where they shouldn't be.
Limited Duration: We designed natural DNA loss over time to trigger bacterial death through our addiction system, combined with normal lung cell turnover. This means our treatment doesn't last forever in patients.
Processing Stability:We use thermal equilibration at 37°C, trehalose protection during freeze-drying, EDTA chelation preventing early activation of our safety systems, then controlled removal and cold storage to maintain our product integrity.
Waste Management:Our lab protocol includes autoclave sterilization of all materials, disinfectant treatment before disposal, specialized containers for hazardous materials, and professional handling of biological waste. We ensure nothing dangerous leaves our facility.
Documentation:We submitted complete iGEM safety forms, detailed check-ins for our non-standard components (our PemK/I system, our CO-BERA RNA, our LLO-L461T), and maintain institutional safety committee oversight of everything we do.
Standards:Our team adheres to NIH guidelines for genetic modification, FDA GRAS criteria, EFSA probiotic standards, and WHO lab safety protocols, with frequent compliance checks. We ensure that our work meets all regulatory criteria.
This multi-layered approach protects through redundant molecular safeguards, environmental controls, and regulatory oversight that our team developed from lab work through patient treatment.
Our team is aware that, despite being intended for therapeutic application, the advanced synthetic biology methods we created for PRESS may be abused for negative purposes. A number of dual-use issues that need careful thought and mitigation techniques have been discovered.
Engineered Bacterial Delivery Systems:Our modified L. plantarum with enhanced targeting capabilities could theoretically be repurposed to deliver harmful substances instead of therapeutic ones. We address this by using GRAS-status organisms that are naturally non-pathogenic, implementing multiple biocontainment systems (PemK-PemI addiction modules, environmental sensors), and ensuring our modifications only function in specific therapeutic contexts (pH <6.9, specific phosphate and temperature conditions).
RNA Interference Technology (CO-BERA):The siRNA delivery system we developed to target TSLP could potentially be modified to silence other essential genes, causing cellular damage or death. We mitigate this through computational screening using siDirect 2.0 to ensure specificity, selective packaging mechanisms that only load intended RNA sequences, and dose optimization that maintains therapeutic efficacy while minimizing potential for misuse.
Modified Membrane-Permeabilizing Proteins: Our LLO-L461T system, designed for controlled endosomal escape, could theoretically be modified back to its more toxic wild-type form or applied to create cell-damaging agents. We address this by maintaining the 100-fold toxicity reduction through the L461T mutation, implementing dual-promoter conditional expression requiring both low pH and high hydrogen peroxide levels, and ensuring expression only occurs in inflammatory environments.
Biocontainment Systems:Paradoxically, our safety systems themselves present dual-use concerns. The PemK-PemI toxin-antitoxin system, while designed for containment, involves toxin production mechanisms that could be studied and potentially misapplied. We mitigate this by selecting systems with no homology to pathogenic toxins, ensuring the system only functions as part of our specific plasmid construct, and requiring multiple environmental conditions for activation.
Information Security:We maintain restricted access to detailed protocols and experimental data, storing sensitive information in secure MySQL databases with authorization limited to essential personnel. Our team shares only necessary information through peer-reviewed publications while withholding specific implementation details that could enable misuse.
Regulatory Oversight:We provide detailed safety documents, including comprehensive check-in forms for all non-whitelisted components, in close collaboration with our Institutional Biosafety Committee and AFCM supervisors
Technical Safeguards: We built multiple redundant safety systems into our design that would be difficult to circumvent without extensive modification. The environmental sensors (temperature, pH, phosphate levels) ensure our bacteria only survive in very specific conditions. The addiction module creates dependency that prevents survival outside controlled conditions. The natural biodegradation of our containment compounds prevents long-term environmental persistence.
Ethical Framework:Our team is dedicated to doing research in an ethical manner, which includes a comprehensive risk assessment at every stage of development, expert input all along the way, and open reporting of safety precautions. We interact with the larger synthetic biology community to share proven safety methods and add to frameworks for risk assessment.
Education and Awareness:Our team members undergo training to know about dual-use awareness, and we create a culture in which everyone is responsible for detecting and reporting usage. We take part in debates regarding the ethical advancement of biotechnology and help develop best standards for the field.
To enhance our safety, we submitted comprehensive iGEM safety documentation including our final safety form covering BSL-1 laboratory work with engineered L. plantarum for asthma therapy, plus three detailed check-in forms for parts not on the White List: a PemK/I toxin-antitoxin biocontainment system, CO-BERA siRNA targeting human TSLP, and modified listeriolysin O (LLO-L461T) for endosomal escape, all with extensive risk assessments and multi-layered safety controls under expert biosafety oversight.
We recognize that our innovations must be developed with consideration for various international settings and cultural contexts. Our cooperative strategy maintains open communication with safety specialists globally and contributes to establishing recognized biosafety knowledge that can guide future research. We actively contribute to the wider synthetic biology community by sharing safe practices that have been shown to be effective while keeping an eye out for information that might allow for dangerous uses.
Our strategy creates an equilibrium between security considerations and scientific advancement, guaranteeing that the advantages of our PRESS asthma treatment can be experienced while lowering the possibility of abuse. As our project progresses and as new possible applications or issues arise, we are still dedicated to regularly reviewing and enhancing our dual-use risk management.
The PRESS project makes a massive shift in asthma management, moving from symptom-based treatments (e.g., corticosteroids, bronchodilators) to specifically genetically engineered probiotic-based therapies that target the inflammatory pathways, such as thymic stromal lymphopoietin (TSLP) mRNA targeting via CO-BERA in the PRESS project. Our long-term goal is developing a safe, and an easily accessible probiotic-based therapy for specific treatment of inflammatory lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), and allergic rhinitis [1, 2]. Our bacteria, which is L. plantarum, can be delivered to the respiratory tissues by a dry powder inhaler (DPI), offering a non-invasive alternative to systemic drugs with fewer side effects [3, 4]. However, there are some other inhaler-based therapies, such as corticosteroids, bronchodilators, our alternative has a great impact more than them, as our therapy doesn't have side effects like the corticosteroids, which could cause candidiasis, and PRESS has a long half-life comparing with these alternatives, decreasing the times of the needed doses with a massive scale.
To transition PRESS from laboratory to clinical field, we made successful experiments in the WI-38 cell line, we plan to conduct preclinical studies in animal models (e.g., ovalbumin-induced asthma in mice) to validate CO-BERA's efficacy in silencing the TSLP-mRNA, reducing TSLP-driven inflammation and airway hyper-reactivity [5, 6]. These studies could assess our treatment's delivery efficiency via the intranasal administration, biodistribution of L. plantarum, and long-term safety of the TA system in a real animal [7]. We should share with some important regulatory bodies, such as the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA), as it will be important for our PRESS to navigate the approval process for L. plantarum to be used in patients' therapy.
Beyond asthma, the design of PRESS, that uses dual-promoter regulation (pKatA and p170-CP25) with the PemKI TA system, can be used to target other inflammatory or infectious diseases with the same mediator in the pathogenicity, TSLP, and the same environment as pH and hydrogen peroxide. For instance, CO-BERA could be re-engineered to target other mRNAs, which are associated with COPD (e.g., IL-8) or viral infections (e.g., SARS-CoV-2 spike protein) [8, 9]. Our used TA system's environmental sensors (thermosensor and PhoB promoter) could be adapted to sense other cues, such as oxygen levels or microbial metabolites, to expand applications to treat some gut-related disorders like inflammatory bowel disease [10, 11].
To build trust and acceptance to our therapy, which is by using a genetically engineered microbiome, we needed to engage stakeholders, patients, and healthcare providers through workshops, and webinars. These efforts addressed ethical concerns about GMOs and especially about our therapy, including potential misuse of TA systems for bioterrorism [12].
The PRESS project prioritizes sustainability to minimize environmental impact and ensure long-term viability of probiotic-based therapies. Our approach integrates ecological, economic, and social dimensions, aligning with the United Nations Sustainable Development Goals (SDGs).
The PemKI TA system, coupled with thermosensor and PhoB promoter, ensures robust biocontainment, preventing engineered L. plantarum from persisting in the environment, outside controlled laboratory settings, or transferring synthetic genes horizontally [13, 14]. To further enhance ecological safety, we will explore auxotrophic strains of L. plantarum that depend on host-specific nutrients, reducing survival outside the lung [15]. Life cycle assessments (LCAs) will be conducted to evaluate the environmental footprint of producing and disposing of PRESS therapeutics, including inhaler devices or lyophilized probiotics. We aim to use biodegradable materials for the delivery system and optimize fermentation processes to reduce energy consumption and waste [16, 17]. Furthermore, our chosen bacterium, L. plantarum, has the ability to break down pesticides and lower methane emissions corresponds with environmental sustainability and may help reduce agricultural pollution while production rises [18].
Our approach integrates SDG 1 (No poverty). To ensure affordability and expansion, we will optimize bioprocessing techniques for L. plantarum fermentation, Lowering expenses by utilizing its metabolic flexibility [19]. Partnerships with global health organizations and biotech firms will support large-scale production and distribution, particularly in low- and middle-income countries where asthma prevalence is rising [20]. We can accelerate genetic engineering by utilizing plasmid-based systems that are compatible with iGEM BioBrick standards, which lowers development costs and permits quick repetitions of therapeutic constructions [21]. In order to finance additional research and ensure economic viability, intellectual property policies will maintain a regulated open-source sharing, which is consistent with iGEM's philosophy, with commercialization [22].
The PRESS project is dedicated to promote equal and community engagement. We will collaborate with local health systems to integrate PRESS into existing asthma care frameworks, training healthcare workers on probiotic administration and monitoring [23]. Campaigns for public awareness will combat the stigma associated with GMOs by highlighting L. plantarum's GRAS status and the TA system's several layers of safety [24, 13]. In order to promote inclusivity, we will create educational materials in multiple languages and involve diverse communities in co-creating implementation strategies, ensuring cultural sensitivity and accessibility [25]. Social impacts, like lower healthcare costs and better quality of life for asthma patients, will be evaluated through long-term patient outcome monitoring [26].
To enhance sustainability, we plan to investigate alternative containment systems, such as CRISPR-based kill switches or synthetic auxotrophies, to complement the TA module [27]. Computational modeling will optimize promoter dynamics and sensor specificity, reducing energy costs in bacterial gene expression [28]. In order to maintain lung homeostasis and make sure that PRESS does not interfere with native microbial communities, we will also investigate formulations that are microbiome-friendly [29]. To meet the objectives of global sustainability, these efforts will be reinforced by integrated partnerships with ethicists, environmental scientists, and politicians.
We hope to transform respiratory disease management while minimizing ecological and social risks, thus, we integrated effective biocontainment (PemK-PemI, thermosensor, PhoB) and sustainable practices. Beyond the lab, PRESS will engage communities, regulators, and industries to ensure ethical, accessible, and environmentally responsible innovation, paving the way for next-generation probiotic therapies, and easing it for other iGEM teams. The long-term vision of our PRESS project is to establish a safe, effective, and expandable platform for microbial therapeutics, with asthma as a proof-of-concept.
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