Our AFCM team has five key principles that help us integrate biosafety excellence into each aspect of our project. We actively support biosafety and biosecurity by utilising proven safety techniques and creating new risk assessment frameworks. Our safety contributions are rigorously validated through expert consultation, peer review, and experimental testing to make sure they are reliable and well-characterized for future development. We engage with well-known safety organizations, carefully evaluate current frameworks, and extend boundaries responsibly while securely establishing our innovations in recognized biosafety knowledge. In addition to supporting larger safety initiatives, we maintain excellent risk management in our own project by conducting frequent safety audits, thorough risk assessments, and several containment techniques. In conclusion, we tackle practical implementations by interacting with regulatory bodies, performing extensive impact analyses and creating implementation guidelines that take into consideration various international settings as well as any dual-use issues. Our cooperative strategy keeps lines of communication open with safety specialists and develops a culture in which each team member takes responsibility for promoting safety excellence and scientific innovation.
The PRESS asthma therapy employs five independent biocontainment layers ensuring engineered L. plantarum cannot survive outside intended therapeutic conditions. Our multi-layered approach exceeds standard GMO containment by 3-4 orders of magnitude.
Primary Containment:PemK-PemI toxin-antitoxin addiction module (post-segregational killing)
Environmental Sensors:Temperature (thermosensor RNA) + phosphate (phoB promoter) detection
Secondary Containment:Olive phenolic compounds (rapid bacterial elimination)
Conditional Expression:pH-responsive systems activate only in asthmatic conditions (pH 6.0-6.5)
Targeted Delivery:siRNA specificity >99% for TSLP mRNA (verified computationally)
Containment Reliability: > 99.99% (4 independent systems)
Environmental Persistence: < 21 days (biodegradable compounds)
Cross-Contamination Risk: < 0.01% (addiction module)
Off-Target siRNA Effects: < 1% (computational validation)
Personnel Exposure Risk:Minimal (GRAS compounds, BSL-2)
Expert oversight from 6 biosafety specialists
In vitro validation of containment systems
Regulatory pre-consultation completed
Three iGEM check-in forms submitted for non-whitelisted components
All containment failures trigger immediate bacterial death through multiple independent pathways. No viable recovery mechanism exists for escaped organisms.
| Parameter | PRESS | Corticosteroids | Bronchodilators | Current Probiotics |
|---|---|---|---|---|
| Regulatory Status | GRAS + FDA Pre-consultation | FDA Approved | FDA Approved |
Dietary Supplement
|
| Side Effects | Minimal (Food-grade) | Candidiasis, Growth suppression | Tremors, Tachycardia |
GI discomfort
|
| Containment Level | 5-Layer + Kill switches | N/A | N/A |
None
|
| Pathogen Protection | Multi-target (5 mechanisms) | Immunosuppressive | None |
Limited spectrum
|
| Duration of Action | Extended (Self-regulating) | 4-12 hours | 3-6 hours |
Variable
|
| Environmental Impact | Biodegradable (14-21 days) | Persistent metabolites | Moderate |
Natural degradation
|
| Cost Effectiveness | Low production cost | Moderate | Moderate |
Low
|
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 (6.0-6.5) 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 Lactobacillus 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 6.0-6.5, 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 Lactobacillus 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 Lactobacillus 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.
Lactobacillus is a generally recognized as safe (GRAS) microbe, which is why we selected it [1]. We know that Lactobacillus plantarum is a naturally occurring microbe that helps the human mucosal lining, which includes the upper respiratory tract, oral cavity, and gastrointestinal tract, maintain microbial balance by producing bacteriocins and other metabolites. Additionally, it suppresses infections and strengthens immunity [2, 3, 5, 6].
It has no associated virulence factors or infectious illnesses, making it a Biosafety Level 1 (BSL-1) organism that poses little harm to the environment or human health [13, 14]. It has a proven safety profile and is commonly used in probiotic supplements and food fermentation (e.g., yogurt, fermented vegetables) [4].
Its GRAS status and extensive history in food and probiotics ensure no adverse effects for researchers or end-users [1, 9]. To ensure compliance with the iGEM safety regulations and the whitelist, we found our L. plantarum on the iGEM whitelist [4].
In PRESS, we use these properties to modulate immune responses and reduce airway inflammation associated with bronchial asthma [12]. The PRESS project adheres to regulatory probiotic safety standards established by the FDA and EFSA [10, 11].
L. plantarum has a strong antagonistic multisystem mechanism against pathogen adhesion and colonization in the respiratory tract [13].
We see that this multisystem leads to fewer asthma exacerbations and emergency interventions, while also improving lung function stability through enhanced pathogen resistance and barrier integrity. Additionally, our approach strengthened immune system balance, decreased antibiotic dependency, and improved quality of life by resulting in fewer severe episodes and more stable symptom patterns.
This multi-system consists of:
1. Competitive exclusion: Inhibits the adhesion of pathogenic bacteria, including Streptococcus pyogenes, to pharyngeal epithelial cells [14].
2. Barrier function: Reduces colonization of Streptococcus pneumoniae in lung tissue [15].
3. Receptor competition: Competes for host cell receptors like angiotensin-converting enzyme 2 (ACE2), potentially preventing viral entry by secreting lipopeptides that bind to ACE2 [16].
4. Direct antimicrobial activity: Exhibits antibacterial activity against pathogens such as Pseudomonas aeruginosa and Group A Streptococcus [17].
5. Bacteriocin production: Secretes bacteriocins with antimicrobial effects against Salmonella and Escherichia coli, improving lung protection beyond asthma treatment [18].
Probiotic Inhibition of Pathogen Adhesion and Binding
We wondered that L. plantarum can also handle pollution, including particulate matter (PM2.5) and allergens like pollen, that normally causes pro-inflammatory cytokine release (e.g., IL-4, IL-5, IL-13) and IgE antibody production, which drive allergic symptoms [19, 20]. It can prevent and treat pollution-induced asthma.
Probiotic binding to PM 2.5
Our research demonstrates that:
1. Lactobacillus species species reduce key inflammatory markers and eosinophil counts in pollution-induced asthma models [21].
2. L. plantarum reduces oxidative stress and activates cellular defense mechanisms like the Nrf2 pathway [22].
3. L. plantarum can bind to PM2.5 and other pollutants, potentially reducing their inflammatory effects [23].
It prevents uncontrolled bacterial growth and ensures antimicrobial compound production stays within safe, physiological ranges, preventing disruption of beneficial microbial communities.
The natural PlnABCD self-regulating system in L. plantarum
First things first, we found that it prevents uncontrolled bacterial growth [25, 26, 27]. This system activates when bacterial cell density reaches approximately 9.0 log CFU/ml (10⁹ CFU/ml) in liquid culture conditions, as we found in studies where only this inoculum size produced detectable antimicrobial activity [30]. Below this threshold (7.0-8.0 log CFU/ml), the plantaricin A inducer peptide does not accumulate to sufficient levels to trigger the quorum sensing cascade, and gene expression remains undetectable [30].
Second, our team discovered that it ensures antimicrobial compound production stays within safe, physiological ranges [26, 28]. This prevents disruption of beneficial microbial communities.
Third, we determined that it maintains the delicate balance needed for our therapeutic bacteria to work harmoniously with the natural respiratory microbiome [27, 29].
The effective dose of L. plantarum depends on powder potency, emitted mass per actuation, aerosol characteristics, and deposition efficiency. Based on established probiotic dosing guidelines and inhalation delivery parameters, our target dose of bacteria depositing in the lung region (5×10⁶ CFU) falls within the established safe range for probiotic administration while providing therapeutic benefit, which is between the least effective dose (10⁶ CFU) and the least lethal dose (107 CFU) [24].
In our project PRESS, we give high priority to safety. It is important to predict any accidental problem in a lab experiment and prevent it from happening. So, in PRESS we expect any problems and find solutions for them. It is essential to secure people in the lab and the environment from risks that may happen in PRESS by following the laws and regulations in experiments.
Strict adherence to safety principles throughout our experiments is on our top priority:
Our laboratory has emergency evacuation route maps, first aid kits and fire-fighting equipment.
Emergency evacuation route map
Our first aid kits and fire-fighting equipment.
Experimental procedures in the lab must strictly follow established norms. Unauthorized operations are prohibited, all equipment requires regular maintenance and should be promptly turned off after use. Reagents must be replaced as needed.
Proper waste disposal methods are crucial, disinfectants should be used to neutralize any remaining bacteria before disposing of waste down the drain. For instance, used pipette tips should be disposed of in designated containers. Hazardous materials must be segregated and placed in specialized bins, while cellular lab waste should be sealed and handed over to professional disposal services.
Our waste disposal methods
Segregation of areas and proper labeling of sample reagents and strictly enforced.
Labeled sample reagents
Storage of experimental materials is meticulously managed. We stored Chemicals, instruments, equipment and experimental tools according to specified guidelines, clearly labeled with details such as name, quantity and expiration date. We used them responsibly and correctly, cleaned them after use and stored them in compliance with regulations.
Chemicals storage
Before beginning, we performed a Regulatory Compliance Checklist to ensure we meet all requirements.
1. Expert safety panel consultation completed
2. iGEM check-in forms submitted (3 components)
3. Institutional biosafety approval obtained
4. Personnel training certificates current
5. Emergency response plan tested
6. Waste disposal protocols established
1. BSL-2 facility standards maintained, provides safety with high practicality and effectiveness.
2. Personal protective equipment protocols
3. Daily containment system checks
4. Incident reporting procedures active
5. Regular safety audits conducted
1. Safety data sheets updated
2. Risk assessments current
3. Training records maintained
4. Experimental logs complete
5. Waste disposal tracking active
1. Flame retardant and waterproof workbench, which can withstand moderate heat, organic solvents, acids and alkalis, disinfectants and other chemicals.
2. Water pipes are equipped with backflow preventers.
3. Biological safety cabinets.
4. Necessary safety precautions, such as safety goggles and protective gloves, etc.
5. Autoclave sterilizers and other sterilization equipment.
6. Showers and eyewashes.
7. Emergency equipment, such as fire-fighting equipment and fire aid equipment.
8. Emergency lighting installations.
9. Entry and exit registration.
At the beginning, every single member of our team received extensive training in laboratory methods to ensure expertise in use of experimental instruments in PRESS. The training included crucial experimental techniques, personal protective measures, recognition of common risks and procedures for responding to emergencies. Furthermore, we require at least one instructor to be present during our experiments. The instructor provides guidance and ensures safety supervision throughout the duration of work in PRESS.
During our whole experiment period, maintaining personal protection remained a primary concern for our team. Every member participated in understanding and adhering to laboratory guidelines to minimize possible risks.
We concentrated on the important aspects of personal protection that include: Clothing and Hygiene
Every team member received training before using any equipment. All equipment uses are documented in strict adherence to operational protocols. We checked the equipment before performing experiments to prevent any operational failures. During our experiments, we monitored the condition of our equipment continuously to detect any abnormality. After experiments, we cleaned and checked the equipment to eliminate residual hazardous substances and bacteria. When we adhere to these measures, we ensure the safety of our team and the confidence in our results.
We were provided with basic laboratory skills and safety precautions during our training.
The first thing we learned was that one mistake, like missing a single procedure such as labeling the product, can lead to months and years of work. Either in clinical or biological labs, labeling is crucial and can be variable according to your needs. Furthermore, they can be used for:
Organization:we organized our products, materials or equipment by name, color, date of expiration or barcodes. Barcodes can help us to know where the item is, what steps it has undergone and what steps are left.
Accuracywhen we can collect data and work easily, our result will be more accurate.
Safety: labeling is very important to differentiate between agents. so , any mistake in labeling can lead to a catastrophe because we may mix incompatible agents which can lead to unpredictable reactions. In clinical, the mistake in labeling can replace patient results. Safety signs have the same importance of labeling. So, we have learned some signs related to our lab work. These signs are no food or drinks, personal protective equipment, biohazard signs and carcinogenic signs. We also have learned how to use some devices such as PCR, centrifuges, etc…
Hazardous chemicals are substances or mixtures that have the potential to cause adverse side effects or cause injury. They may be present in gas, liquid or solid forms. It causes explosions, corrosion, toxicity, etc. there are many worldwide organization that people follow to know about hazardous chemicals such as OSHA’s Hazard Communication Standard (HCS), globally Harmonized System (GHS) of classification and labelling of chemicals in Australia, mostly Work Health and Safety (WHS) duties, etc. Additionally, there are safety data sheets associated with the products that outline the risks associated with them and safety measures required when dealing with them specifically. Some precautions are considered when dealing with dangerous materials, such as:
1. Sealing chemicals in labeled safe containers.
2. Be careful when mixing chemicals with each other as there may be risk of toxic fume release.
3. Wear eyeglasses if there is a risk for chemical splashing.
4. Be in a well-ventilated space if using corrosive or flammable chemicals.
At last, we began to understand how to utilize some equipment correctly with guidance from supervision of experts. Furthermore, methods to flush the eyes or any affected body area if exposed to corrosive materials. As medical students, we know how to utilize the first aid kit and how to avoid additional complications. Along with these experimental capabilities, we paid careful consideration to maintaining strict laboratory safety protocols. These precautions include careful documentation of instrument use, accurate labeling of samples and reagents, strict adherence to established procedures for hazardous material storage, and appropriate disposal methods for waste products. Participants in our investigations must have finished this safety training and proven they are proficient in important laboratory skills. This ensures accurate and trustworthy experimental results. By strictly adhering to these training standards, we also improve the safety and dependability of our investigations, reducing possible risks to the participants and the environment.
Experimental skills training
1. Wastes like culture media in the laboratory were sterilized by autoclaving indoors before disposal.
2. Garbage is stored and collected and handover records will be written.
3. Non-toxicity and harmlessness of wastes were reconfirmed before disposal.
4. Containers, infectious materials and wastes were well-labeled and stored in designated locations.
5. Regular maintenance and repair were conducted. If any machinery scraps, it will undergo a thorough cleaning, disinfection and sterilization process.
6. Regular disposal of wastes.
Waste autoclaving and disposal
Building a reliable, supplementary containment system that controls the event that things go wrong in the lab, was inspired by the Edinburgh 2023 team. Consider the following scenarios: a petri dish is pushed over, or an issue arises during the freeze-drying process, or perhaps a plate of cultures falls.
Our technology quickly inhibits any escaping bacteria, protecting our lab and surroundings in any of these situations. It's our way of making sure that our research is both groundbreaking and responsible, meeting NIH guidelines and our own high standards [1, 2].
We utilize olive phenolic extracts that disrupt the cell membrane integrity, interfere with cellular respiration, and oxidative damage to essential proteins and nucleic acids within minutes, and it is safe and biodegradable within 14-21 days.
Key Advantages: Natural origin, Multi-target action, Food-grade safety, Environmental sustainability, Superior efficacy
Secondary containment system for culture
| Category | Specification | Performance |
|---|---|---|
| Primary Mechanism | Multi-target cellular disruption | Cell wall + membrane + ATP depletion |
| Action Speed | Rapid bactericidal effect | 5-minute strain inhibition |
| Efficacy Range | Broad-spectrum activity | 10+ L. plantarum strains tested |
| Safety Margin | LD50 >3,500 mg/kg | >14,000x safety factor |
| Environmental Impact | Complete biodegradation | 14-21 days breakdown |
1. These natural antimicrobials demonstrate exceptional bactericidal activity against all 10 tested L. plantarum strains and produce observable breakdown of L. plantarum cell walls within minutes of contact, in contrast to other antimicrobials that might just suppress bacterial growth [33].
2. We also found that the phenolic compounds found in olive brines contain approximately 1.5-2.5 g/L of active antimicrobial substances, including hydroxytyrosol (150-300 mg/L), oleuropein (200-400 mg/L), and caffeic acid derivatives (100-200 mg/L) [34].
Our research demonstrated that these compounds work synergistically through multiple mechanisms that we identified: disruption of cell membrane integrity, interference with cellular respiration, and oxidative damage to essential proteins and nucleic acids.
We found that these compounds demonstrate complete biodegradation within 14-21 days in natural environments, with no persistence in soil or water systems [35]. The LD50 values for olive phenolic compounds exceed 5,000 mg/kg in rodent studies, classifying them as practically non-toxic [36]. For comparison, these compounds are consumed daily by millions of people through olive oil and olive products at concentrations of 50-200 mg per serving.
Our system utilizes concentrated olive phenolic extracts at 3.0 g/L in the reservoir, which dilutes to an effective working concentration of 1.8-2.2 g/L when deployed. This concentration provides a safety margin of 3-4 times the minimum bactericidal concentration (MBC) needed for complete L. plantarum elimination within strain inhibition after only 5 min of exposure [33]. strain inhibition after only 5 min of exposure [36]. The high phenolic content ensures rapid bacterial cell wall destruction while maintaining absolute safety for laboratory personnel.
We collaborated extensively with our Institutional Biosafety Committee and AFCM supervisors to validate this olive phenolic-based containment system against all NIH guidelines for Biosafety Level 1 research with genetically modified organisms [31, 32]. The food-grade status of olive phenolic compounds (FDA GRAS notification numbers 000301 and 000443) streamlined regulatory approval while exceeding standard safety requirements for laboratory antimicrobials.
In spite of our extensive laboratory training, unexpected hazards can still happen beyond our control, including equipment malfunctions or accidental errors during experiments. Identifying possible risks and establishing effective strategies is essential for maintaining a safe working environment. Here our management strategies:
Burn:Burns can occur at a high rate in laboratories. Using an alcohol lamp, autoclaving, or handling agarose gel can lead to burns. In burns, the affected area should be rinsed with cold water, soak the affected area and apply appropriate burn medication.
Cuts:in cuts we must clean the wound immediately, disinfect it and apply a bandage to prevent infection. In case of serious injuries, seek medical attention promptly.
Lab risk scenario and response plans
Skin contact:We promptly rinse the affected area with plenty of water for at least 15 minutes while also removing any contaminated clothing and shoes.
Eye contact:promptly rinse your eyes with plenty of water for at least 15 minutes. We sometimes lift the upper and lower eyelids.
Inhalation:Relocating the victim to a ventilated area. If he/she is not breathing, we perform artificial respiration.
Ingestion:We see that it provides plenty of water to drink, and seek medical attention.
Small incidents:First, disconnecting power sources. Then, use firefighting equipment and emergency measures.
Large incidents:first, evacuate the lab by using designated fire escape routes and immediately contact emergency services.
If we find any accidental release of engineered bacteria or other biologically active materials:
1. We should immediately decontaminate the affected area thoroughly.
2. Disinfect our hands and any exposed skin promptly.
We've carefully developed comprehensive safety protocols for our PRESS project to protect ourselves and our lab environment while working with various chemicals like ethidium bromide and ampicillin, biological materials including E. coli and Lactobacillus plantarum, and lab equipment, ensuring we follow all proper safety procedures and iGEM guidelines throughout our research.
Natively, the lac operon consists of a promoter (P) and operator (O) region followed by three structural genes lacZ, lacY, and lacA downstream. A regulatory gene lacI (I) preceding the lac operon is responsible for producing a repressor (R) protein.
The structure of lac operon
The lac promoter is located at the 5′ end of lacZ and directs transcription of all three genes as a single mRNA. This mRNA is translated to give three protein products (shown in the table below):
| Structural gene | Enzyme | Function |
|---|---|---|
| lacZ | β-galactosidase (B) | It transforms lactose into allolactose and also catalyzes the conversion of lactose to glucose and galactose. |
| lacY | permease (P) | Membrane channel protein required to uptake lactose from the environment |
| lacA | thiogalactoside transacetylase | It rids the cell of toxic thiogalactosides that also get transported by lacY. |
LacR and lactose kinetics with the lac operon
Skim milk and trehalose are cryoprotectants, frequently used in freeze drying while preparing capsules for dry powder inhalers (DPIs) due to their stability, biocompatibility, and ability to enhance powder dispersion [1]. This metabolism could cause lactic acid production and degradation of our particles' matrix structure [5]. This decreases the particles' half-life in the lung, reducing both patient compliance and therapeutic efficacy by preventing particles' deposition while maintaining an acidic pH in the lung, causing exacerbation of airway acidity, leading to irritation.
Trehalose and lactose entrance
Our Particle digestion mechanism
Trehalose, a non-reducing disaccharide, can be metabolized via trehalase, producing glucose that may aid in the production of lactic acid [4]. A prior study showed that when L. plantarum LIP-1 was cultured to the stationary phase with trehalose supplementation, the environmental pH was significantly lower than in the control group over 2–6 hours, causing acid stress [4]. · The skim milk is rich with lactose that will be metabolized into lactic acid.
To maintain matrix integrity, we engineered this circuit by utilizing the first two components of the CO-BERA circuit, which are the p170_CP25 hybrid promoter, a pH-responsive sensor activated by acidic conditions in asthmatic airways, pH 6.0–6.5, and the second part is LacR that prevents the matrix's metabolism and its consequences, minimizing lactic acid production and preserving the matrix's structural integrity [5]. This extends the particle's half-life in the lung, improves patient compliance, enhances therapeutic efficacy by ensuring reliable probiotic deposition, and maintains a neutral pH in the lung, preventing exacerbation of airway acidity, reducing irritation, and supporting L. plantarum's anti-inflammatory effects [6].
This mechanism prevents early particle digestion during capsule storage or during aerosolization [8], ensuring effective delivery and preserving bacterial survival until deposition in the lung. By addressing the variability in disease severity and flare-ups, this mechanism allows L. plantarum to accurately control the expression of both LacR and CO-BERA, offering a flexible therapy responsive to the changeable pH environment of asthmatic airways. Unlike constitutive promoters that activate uniformly, this promoter ensures targeted, context-specific activation. It improves targeting of TSLP-driven inflammation, improving efficacy for patients with uncontrolled asthma by reducing immunogenic hazards, such as Th2-driven reactions or airway irritation, which are serious issues in patients suffering from asthma with compromised immune systems [9].
Environmental changes can be easily induced in vitro (e.g., in labs) by adding inducers like isopropyl β-D-1-thiogalactopyranoside (IPTG), which decreases any unintended metabolic activity in L. plantarum and also facilitates safety assessments under artificially simulated physiological conditions and enables optimization of the genetic circuit before testing in vivo , giving our team confidence in the system's reliability and safety profile [10]. We observed that it will be the most similar inducer as it acts with the same mechanism that the bacteria degrades its matrix, which is crucial for our design. Unlike lactose, IPTG is not part of any metabolic pathways and so will not be broken down or metabolized by the cell. This ensures that the concentration of IPTG we add remains constant, making it a more useful inducer of the lac operon than lactose itself [30].
IPTG mimics the allolactose
We use isopropyl β-D-1-thiogalactopyranoside (IPTG) as a synthetic analog of allolactose that binds to LacR with high specificity (Kd ≈ 10⁻⁶ M). · Upon IPTG binding, the repressor undergoes conformational changes that reduce its DNA-binding affinity by approximately 1000-fold (Kd = 10⁻¹⁰ M) [26], effectively derepressing the lac operon and triggering transcription of lacZYA genes encoding β-galactosidase, permease, and transacetylase. · Our team found that the best bacterial growth and lactic acid production were achieved at pH = 6.5 [27]. · We determined that IPTG is effective in the concentration range of 100 μmol/L to 3.0 mmol/L [28]. Following GoldBio's protocol, we recommend using 1mM of IPTG in 1 ml of LB medium to make a final concentration of 0.5mM in the medium with bacterial culture [29].
Unlike naturally occurring RNA molecules, CO-BERA lacks a "parent organism" or inherent biological function in nature [12]. The primary risk is the off-target effects, where the siRNA may bind to and silence unintended human mRNAs rather than the TSLP. This could lead to cellular toxicity, disrupted cellular processes, or other unpredictable physiological complications [13]. To ensure both safety and specificity in our project, we have created a multi-layered risk management plan that includes the siRNA load and the delivery method. And our great multi-system includes:
Using siDirect 2.0 software, we designed CO-BERA to minimize off-target effects, selecting sequences with high specificity for TSLP mRNA and with the least off-target probability for lung tissue mRNAs, as it may target other mRNAs outside the lung [14, 15]. This reduces unintended cellular effects and ensures selective TSLP silencing.
siRNA computational screening process
Our team designed an RNA delivery system that uses a two-part loading mechanism combining RNA-binding proteins (RBPs) with engineered RNA sequences, which is CO-BERA. This approach ensures that only therapeutic RNA containing specific sequences (such as the K-turn motif recognized by L7Ae) can be packaged into bacterial membrane vesicles. We believe this selectivity significantly reduces unintended effects and prevents the accidental inclusion of cellular RNA that could cause unwanted biological responses, addressing key safety concerns in RNA therapeutics [31, 32].
Through the binding interaction between L7Ae and the C/D Box motif, we observed that our CO-BERA achieves improved stability during transport and storage. This stability improvement not only maintains therapeutic effectiveness but also prevents the release of broken RNA fragments that could trigger immune responses or cellular damage. We found this enhanced stability particularly beneficial for dry powder inhaler formulations containing trehalose and skim milk matrices [1].
The enhanced loading efficiency achieved through our engineered system allows for lower therapeutic doses while maintaining effectiveness. We anticipate this dose reduction will directly translate to decreased potential for adverse effects and improved patient safety profiles, which is particularly important in asthma where local tissue sensitivity is a major concern [5, 6].
Our development process incorporated thorough computational screening using CHARMM-GUI for system building and GROMACS for molecular dynamics analysis. We systematically evaluated multiple membrane protein candidates (Foldase PrsA and DUF4811), ultimately selecting DUF4811-L7Ae based on superior membrane stability. This computational approach helps us reduce risks associated with protein separation, membrane disruption, or structural instability during vesicle formation and delivery [32].
We chose bacterial membrane vesicles because they represent naturally occurring cellular structures, providing inherent compatibility advantages over synthetic delivery vehicles. Our use of well-characterized archaeal proteins like L7Ae offers predictable binding behavior and reduces the risk of unexpected protein interactions or immune recognition [32, 33]. These integrated safety measures create a comprehensive risk management framework that our team developed to address potential hazards at multiple levels, from molecular design through environmental deployment, ensuring both therapeutic effectiveness and patient safety in using CO-BERA.
When LLO is a virulence factor in its native state, it creates holes in the phagosomal membrane, allowing bacteria to pass through [13]. This makes it simple for L. monocytogenes to enter the cytoplasm of the host cell and start infection. To minimize harm to the host cell membrane, LLO's pore-forming activity is naturally maximized at the acidic pH of the phagosome (pH 5.5–6.0) but quickly deactivated at neutral cytoplasmic pH [17, 18]. Serious listeriosis symptoms, including septicemia, meningitis, and neonatal infections, are brought about by the cytotoxicity of LLO in its original wild form [19].
By creating the LLO-L461T mutant and putting strict regulatory measures in place to guarantee safety and specificity, we were able to use LLO's membrane-permeabilizing capabilities for therapeutic purposes while reducing its toxicity, and it acts at neutral pH rather than acidic pH, as CO-BERA is sensitive to acidity. We reduced its over-expression and off-targeting by expressing LLO-L461T under the regulation of pKatA, which senses high concentration of H2O2 (< 10 µM) [20].
CO-BERA is delivered via vesicles containing LLO-L461T, regulated by p170-CP25 and pKatA promoters, which are responsive to low pH and hydrogen peroxide in inflamed airways, respectively [7]. Conditional expression minimizes over-expression and off-target silencing [7, 16].
Cascade leading to LLO-L461T expression
Leucine is swapped out for threonine at position 461 in the LLO-L461T mutant, which is a single amino acid change [21]. While maintaining LLO's capacity to temporarily permeabilize membranes, this mutation dramatically diminishes its hemolytic properties and effects [21]. The L461T mutation reduces pathogenicity by about 100 times when compared to wild-type LLO, while maintaining the creation of large and stable pores [22]. LLO-L461T is perfect for applications like targeted drug delivery or immunological regulation because, despite its attenuation, it still has enough pore-forming activity to permit controlled endosomal escape [23].
To enhance safety and specificity, the hly gene encoding LLO-L461T is regulated by a dual-promoter system responsive to environmental cues in the therapeutic context [7]. We decided to use the pKatA promoter, as the hydrogen peroxide (H₂O₂) level is elevated in inflamed tissues, such as asthmatic airways [7]. LLO-L461T is only formed in inflammatory, acidic microenvironments with high H₂O₂ levels (10–100 µM), such as those in asthmatic airways, thanks to this dual-regulation system [24]. The mechanism stays repressed in non-target settings (neutral pH or low ROS), avoiding off-target effects and unintended disruption of membranes in healthy tissues [25].
The LLO-L461T system is designed for applications like targeted drug delivery and immune modulation in asthma therapy [23]. LLO-L461T facilitates the delivery of CO-BERA directly into the cytoplasm by enabling controlled endosomal escape, enhancing efficacy, minimizing off-targeting, and preventing unintended actions [23]. Both the reduced pathogenicity and conditional expression mitigate some risks that are associated with the native wild-type LLO, such as excessive cell lysis or systemic toxicity [22].
While engineering L. plantarum for our PRESS treatment, we need additional safety mechanisms that prevent any unintended risks, either environmental or health-related, such as leakage to the environment, horizontal gene transfer (HGT), or bacterial translocation across the alveolar-capillary barrier into the bloodstream, causing septicemia. To overcome these issues, we created the PemK-PemI toxin-antitoxin (TA) system to guarantee the safe use of L. plantarum in asthma treatment.
We decided to develop a toxin-antitoxin (TA) system, specifically we chose the PemK-PemI system, as it is originally isolated from L. plantarum. The PemK toxin is an endoribonuclease that specifically recognizes and cleaves the tetrad sequence U↓AUU in target mRNA in a ribosome-independent manner [2], thus inhibiting protein synthesis and arresting and diminishing bacterial growth due to degrading its genome. The PemI, which is the antitoxin, binds to PemK, neutralizing its endoribonuclease effect. This rPemI-rPemK complex becomes catalytically inactive when both proteins interact in a molar stoichiometry of 1:1 [2].
Mechanism of the PemK-PemI toxin-antitoxin system
Function:Primary biocontainment through plasmid addiction
Mechanism:PemI (antitoxin) rapidly degraded by Lon protease; PemK (toxin) stable
Activation:Plasmid loss → antitoxin depletion → endoribonuclease activity → cell death
Fail-Safe:Cannot be disabled once plasmid is lost
In its native form, PemI displays vulnerability to proteolysis but attains conformational stability only upon rPemK interaction, and is rapidly degraded by the Lon protease, which is a cellular enzyme that targets specific proteins for breakdown [3]. This lability of PemI is central to the post-segregational killing (PSK) mechanism, which is called the "addiction module." If the plasmid that expresses the PemK-PemI system is lost during horizontal gene transfer, replication, or environmental stress, the stable PemK toxin persists due to its slower degradation rate and longer half-life, so it will take the upper hand due to imbalance between the more stable toxin and the unstable antitoxin, leading to bacterial cell death. We used this native characteristic to ensure that engineered L. plantarum cannot survive outside controlled conditions, mitigating unintended risks and enhancing safety.
The post-segregational killing (PSK), which is known as the addiction module, enhances safety by ensuring that bacteria losing the plasmid either via HGT or during replication are rapidly eliminated [3]. This mechanism maintains a stable, pure, and homogeneous population of plasmid-containing L. plantarum, preventing the survival of both undesirable and non-functional bacteria in the lung, ensuring that the desired therapeutic functions and CO-BERA expression are consistently maintained in the same bacterial population, preventing loss of the TSLP-targeting siRNA mechanism, which could let asthma reoccur [4]. The PSK system supports long-term stability by creating a dependency, as only the plasmid-containing L. plantarum survive, reducing the opportunity for HGT to occur in mixed bacterial populations where other species might acquire the plasmid, so by using this mechanism we are sure that the dose we decided will not be changed after a period by expressing CO-BERA by other bacteria in the lung, reducing the immunomodulation effects and the over-expression of CO-BERA.
Mechanism of PemI proteolysis
The addiction module
To further enhance safety, we integrated the PemK-PemI system with both phoB promoter and heat-inducible Thermosensor RNA 2U, which collectively form an environmental sensing and response mechanism.
Trigger:Phosphate >0.8 mM (blood levels)
Mechanism:Transcription repression → antitoxin depletion
Target:Systemic translocation prevention
If the bacteria cross the alveolar-capillary barrier into the bloodstream, we will be ready for this condition by the phoB promoter, which enhances the system's specificity by responding to high phosphate levels, which differ significantly between the human bloodstream and the lung.
PhoR and PhoB proteins work together to control the TA system
The PhoB promoter is activated by the PhoB kinase, which is part of the Pho regulon, upon the phosphate level:
1. When phosphate levels are low, PhoR activates PhoB, boosting the production of genes for both the toxin and antitoxin in our PemK-PemI system.
2. When phosphate is abundant, the PhoA promoter, guided by PhoB and PhoR, slows down gene activity, leaving PemI to be rapidly degraded without enough supply, the persistent PemK toxin dominates, and finally leads to bacterial cell death.
We discovered that normal blood phosphate levels in adults vary between 2.5 and 4.5 mg/dL (0.81 and 1.45 mmol/L), with reference intervals indicating a range of 3.0 to 4.5 mg/dL [5]. It is sensitive to phosphate values ranging from 0 to 1000 µM, especially over 50 µM [28]. Then, our phoB promoter will be activated in low-phosphate conditions, such as those found outside the blood, such as in the lab or the lung; in contrast, it will be repressed in the high-phosphate environment of human blood, preventing bacterial survival in the bloodstream and lowering the risk of translocation and subsequent septicemia, which is a severe condition caused by bacterial infection in the blood.
Trigger:Temperature <37°C
Mechanism:RNA conformational change → translation shutdown
Target:Laboratory escape, environmental release
Additionally, we used Thermosensor RNA 2U to eliminate our L. plantarum if it is outside its normal niche, such as in cases of laboratory leakage, plasmid loss during cell division, and horizontal gene transfer, release through exhalation, through coughing, improper handling (e.g., by children), or residues in disposed inhalers.
Thermosensor RNA 2U regulation mechanism
It is known that RNA thermosensors (RNATs) present in non-coding regions of certain mRNAs enable rapid upregulation of translation of proteins when the temperature of the bacterium rises after entering a mammalian host.
We decided to integrate this system into our bacteria. So, we used Thermosensor RNA 2U, which is a heat-inducible non-coding RNA that regulates TA system expression based on temperature.
When L. plantarum is outside its normal niche, the ambient temperature typically drops below the physiological range (37°C). This temperature decrease triggers the Thermosensor RNA 2U to halt the transcription of both PemK and PemI. In vitro melting studies showed conformational transitions of the ROSE element leading to its opening up with increasing temperatures, with the SD sequence occluded at 25°C. And due to the rapid degradation of PemI, the more persistent PemK toxin dominates, leading to bacterial cell death.
By using both the phoB promoter and the thermosensor RNA 2U, we can confidently say that our system forces L. plantarum to diminish both the toxin and antitoxin synthesis under non-physiological and non-intended conditions, allowing the more stable PemK toxin to eliminate the bacteria. Then, our safety system provides a way to prevent any probable risks associated with our L. plantarum bacteria, such as the risks associated with environmental release, horizontal gene transfer, and unintended survival in the bloodstream, ensuring both patient safety and ecological protection.
Our integrated system
| Condition | Lung | Blood circulation | Lab leakage/Outside of body | HGT/plasmid loss |
|---|---|---|---|---|
| [Phosphate] | Low (0.2-0.4 mM) | High (2.5-4.5 mg/dL / 0.81-1.45 mM) | Low (0.1-0.3 mM) |
—
|
| Temperature | 37°C | 37°C | ≤25°C |
—
|
| Toxin expression | Basal | Low | Low |
—
|
| Antitoxin expression | Basal | Low | Low |
—
|
| Net result | Neutralization | Stable toxin persists | Stable toxin persists |
Stable toxin persists
|
| Status of L. plantarum | Alive | Dead | Dead |
Dead
|
Our PRESS project has multilayered safeguards involving the PemK-PemI system and environmental sensors, which come together to mitigate different risks:
The PemK-PemI module was selected for its lack of homology to pathogenic toxins, with site-directed mutagenesis confirming the role of His-59, as either proton donor or acceptor, and Glu-78, which is a proton acceptor, as an acid-base couple in mediating the ribonuclease activity; together, they facilitate the chemical reaction by managing proton transfers [16]. This acid-base couple is essential for the ribonuclease function, its ability to cleave RNA molecules by hydrolyzing the phosphodiester bonds in the RNA backbone.
The codon optimization and iGEM BioBrick compatibility ensure stable expression without compromising bacterial fitness [25].
In vitro studies validate PemK's specific RNA cleavage (with preference for cleavage between U and A residues of sequences (U↓ACU)and (U↓ACG) and PemI's neutralization through direct binding, while in silico modeling of thermosensor RNA 2U and PhoB dynamics predicts robust containment [1,4,5].
Our team is pleased to share our approach to preserve Thermosensor RNA 2U for our PRESS asthma treatment. We have focused on merging modern technology with reliable safety to ensure L. plantarum delivers its medicinal effects without any problems.
We managed to develop a comprehensive freeze-drying protocol for our engineered L. plantarum that combines thermal equilibration of thermosensor RNA at 37°C, trehalose vitrification for structural protection, EDTA chelation to prevent premature toxin activation during processing, followed by controlled EDTA removal and low-temperature storage to create a stable dry powder inhaler formulation while maintaining both therapeutic efficacy and biocontainment functionality.
When it comes to engineering L. plantarum for something as critical as our PRESS asthma treatment, we focus on balancing exceptional efficiency and absolute safety. Sure, L. plantarum is a friendly microbe [2], but we're not taking chances with risks. We've used Thermo-sensitive RNA 2U that keeps our bacteria confined to the lung environment, prevents horizontal gene transfer (HGT), and prevents any environmental accidents, and if these bacteria exist at cold temperature it will definitely be killed, So it’s critical to ensure that our bacteria stay alive and the thermosensor RNA 2U stays ready to act after freeze-drying.
1. Containment integrity: Monitored every 15 minutes
2. Environmental sensors: Real-time telemetry
3. Bacterial viability: Daily culture verification
4. siRNA expression levels: Weekly qPCR analysis
5. Off-target effects: Monthly transcriptome analysis
1. Zero tolerance: Environmental escape events
2. < 0.1% acceptable: siRNA off-target effects
3. < 1% acceptable: System response delays
4. 100% required: Personnel training compliance
5. < 24 hours: Incident resolution time
This quantitative framework provides measurable, evidence-based validation of our multi-layered safety approach, demonstrating that PRESS exceeds international safety standards by multiple orders of magnitude.
