We integrated safety throughout our entire project. We chose the most secure organism available, provided comprehensive training to all team members, and fully equipped our laboratory with emergency equipment. We created multiple protection mechanisms: our treatment activates only at the target location, affects only the intended component, and our modified organism survives exclusively in the treatment zone. It self-destructs in all other locations, including oral areas, blood circulation, lab spaces, and external settings. We incorporated additional safeguard systems, completed all necessary regulatory paperwork, developed contingency protocols for all situations, and worked under continuous expert supervision. If any single safety feature fails, several backup systems remain operational to safeguard researchers, patients, and the ecosystem.
We prevent premature matrix degradation through sophisticated control of the Lac operon, using pH-responsive LacR regulation that maintains particle integrity during storage and lung deposition while preventing lactic acid production.
Skim milk is a cryoprotectant frequently used in freeze drying when preparing capsules for dry powder inhalers (DPIs) due to its stability, biocompatibility, and ability to enhance powder dispersion [1]. In our project, we use L. plantarum, which is a lactic acid bacterium that can metabolize lactose into lactic acid.
This metabolism causes lactic acid production and degradation of our particles' matrix structure [5], decreasing the particles' half-life in the lung. This reduces both patient compliance and therapeutic efficacy by preventing particle deposition while maintaining an acidic pH in the lung, causing exacerbation of airway acidity and irritation.
To ensure particle stability, we must prevent premature lactose metabolism. Here's how the natural Lac operon works and how we control it:
Before showing our approach to choosing a perfect design with its safety considerations, we need to introduce you to the Lac operon, which is naturally found in bacteria. The Lac operon consists of a promoter (P) and operator (O) region followed by three structural genes: lacZ, lacY, and lacA located downstream. A regulatory gene called lacI (I), which precedes the Lac operon, is responsible for producing a repressor (R) protein.
We found that the lac promoter is located at the 5′ end of lacZ and directs transcription of all three genes as a single mRNA. We observed that this mRNA is translated to produce three protein products (shown in the table below):
| Structural gene | Enzyme | Function |
|---|---|---|
| lacZ | β-galactosidase (B) | It transforms lactose into allolactose and catalyzes the conversion of lactose to glucose and galactose. |
| lacY | permease (P) | It is a membrane channel protein required for the uptake of lactose from the environment. |
| lacA | thiogalactoside transacetylase | It rids the cell of toxic thiogalactosides that also get transported by lacY |
We found that LacY permease functions to transport lactose and other β-galactosides across the cell membrane, allowing these sugars to enter the bacterial cell, preparing it to be metabolized.
Normally, lacI, which is located prior to the Lac operon, produces a repressor (LacR) protein that inhibits the Lac operon by binding to its operator and stopping the expression of Lac operon proteins. This scenario occurs when lactose is absent from the bacteria. However, if lactose molecules are present in our bacteria, these molecules will transform into allolactose and bind to the LacR, preventing its function and derepressing the Lac operon protein expression.
To maintain matrix integrity and prevent or delay matrix degradation, we used the first two components of the CO-BERA circuit with their secondary function. These components are the p170_CP25 hybrid promoter, which serves as a pH-responsive sensor activated by acidic conditions in asthmatic airways (< pH 6.9), and LacR, which prevents the milk protein matrix's metabolism and its consequences. This approach minimizes lactic acid production and preserves the matrix's structural integrity for a longer time [5]. This strategy extends the particle's half-life during storage and in the lung, improves patient compliance and enhances therapeutic efficacy by ensuring reliable probiotic deposition. Additionally, it 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 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 changing 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, enhancing efficacy for patients with uncontrolled asthma by reducing immunogenic hazards, such as Th2-driven reactions or airway irritation, which are serious issues in asthmatic patients with compromised immune systems [9].
Artificial changes can be easily induced in vitro (e.g., in labs) by adding inducers like isopropyl β-D-1-thiogalactopyranoside (IPTG) along with skim milk to L. plantarum. This approach facilitates safety assessments under artificially simulated real conditions when the bacteria metabolize the lactose in skim milk into lactic acid. IPTG binds to LacR and allows the lactose to be metabolized, which saves us time and enables optimization of the genetic circuit before testing in vivo. This process gives our team confidence in our system's reliability and safety profile [10].
We observed that IPTG will be the most suitable inducer as it acts with the same mechanism, allowing the bacteria to utilize the Lac operon effectively. Unlike lactose, IPTG is not part of any metabolic pathways and will not be broken down or metabolized by the cell. This ensures that the concentration of IPTG we add remains constant throughout the experiment and will not affect the results due to concentration fluctuations, making it the most reliable inducer of the Lac operon [30].
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 lacZ, lacY, and lacA genes encoding β-galactosidase, permease, and transacetylase, respectively. Our team found that optimal 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 adding 1 mM IPTG to LB medium containing the bacterial culture to achieve a final concentration of 0.5 mM [29].
Unlike natural RNA molecules, CO-BERA lacks a "parent organism" or inherent biological function in nature [12]. The primary risk is off-target effects, as the siRNA may bind to and silence unintended human mRNAs rather than 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 encompasses CO-BERA design and delivery method. Our comprehensive multi-system approach includes:
Using siDirect 2.1 software, we designed CO-BERA with minimal off-target effects, selecting sequences with high specificity for TSLP mRNA and minimal off-target probability for lung tissue mRNAs, while avoiding sequences that may target other mRNAs outside the lung [14, 15]. This reduces unintended cellular effects and ensures selective TSLP silencing.
Our team designed a sophisticated RNA delivery system that uses a two-part loading mechanism combining RNA-binding proteins (RBPs) with engineered RNA sequences to create CO-BERA. This approach ensures that only our therapeutic RNA containing specific sequences (such as the K-turn motif recognized by L7Ae) can be packaged into bacterial membrane vesicles.
We believe that this selectivity significantly reduces unintended effects and prevents the accidental inclusion of cellular RNA that could trigger 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 degraded RNA fragments that could trigger immune responses or cellular damage.
We found this enhanced stability is particularly beneficial for dry powder formulations containing trehalose and skim milk matrices [1].
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 helped us reduce risks associated with protein separation, membrane disruption, or structural instability during vesicle formation and delivery [32].
Dive deeper into the details on our model page!"
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 we developed to address potential hazards at multiple levels, from molecular design through environmental deployment, ensuring both therapeutic effectiveness and patient safety when using CO-BERA.
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].
LLO is a virulence factor that, in its native state, creates holes in the phagosomal membrane, allowing bacteria to pass through [13]. This enables L. monocytogenes to easily enter the cytoplasm of the host cell and initiate infection. LLO's pore-forming activity is naturally maximized at the acidic pH of the phagosome (pH 5.5–6.0) but becomes quickly deactivated at neutral cytoplasmic pH [17, 18]. The cytotoxicity of wild-type LLO causes serious listeriosis symptoms, including septicemia, meningitis, and neonatal infections [19].
By creating the LLO-L461T mutant and implementing strict regulatory measures to ensure safety and specificity, we were able to harness LLO's membrane-permeabilizing capabilities for therapeutic purposes while reducing its toxicity and making it act at neutral pH (pH 6.5) rather than acidic pH, since 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 concentrations of H₂O₂ (> 10 µM) [20].
| Feature | Wild-Type LLO | LLO-L461T |
|---|---|---|
| Toxicity | High pathogenicity | 100x reduced |
| pH Optimum | Acidic (pH 5.5-6.0) | Neutral (pH 6.5) |
| Expression | Constitutive | pKatA-regulated (H₂O₂-responsive) |
| Pore Formation | Extensive cell lysis | Controlled endosomal escape |
| Safety | Dangerous | Therapeutic |
In the LLO-L461T mutant, we substituted leucine with threonine at position 461, representing a single amino acid change [21]. While maintaining LLO's capacity to temporarily permeabilize membranes, this mutation dramatically diminishes its hemolytic properties [21]. The L461T mutation reduces pathogenicity by approximately 100-fold compared to wild-type LLO, while maintaining the ability to create large and stable pores [22]. Despite its attenuation, LLO-L461T retains sufficient pore-forming activity to permit controlled endosomal escape, making it ideal for applications such as targeted drug delivery and immunological regulation [23].
The pore-forming function enables our bacteria to escape from phagosomes and neutrophils, which allows the bacteria to persist in inflammatory environments despite phagocytosis, increasing their half-life in the lung and decreasing the need for re-dosing.
To enhance safety and specificity, we regulated the hly gene encoding LLO-L461T using a promoter responsive to environmental cues in the therapeutic context [7]. We chose the pKatA promoter to ensure that LLO-L461T is only expressed in inflammatory microenvironments with high H₂O₂ levels (10–100 µM), since hydrogen peroxide (H₂O₂) levels are elevated in inflamed tissues, such as asthmatic airways [7, 24]. This mechanism remains repressed in non-target settings with low ROS, preventing over-expression and off-target effects while avoiding unintended membrane disruption in healthy tissues [25].
The LLO-L461T system is designed for applications such as 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-target effects, and preventing unintended cellular activity [23]. Both the reduced pathogenicity and conditional expression mitigate risks associated with the native wild-type LLO, such as excessive cell lysis or systemic toxicity [22].
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Lactobacillus is generally recognized as a safe (GRAS) microbe, which is why we selected it [1]. Lactobacillus plantarum is a naturally occurring microbe that supports the human mucosal lining, including the oral cavity, and gastrointestinal tract. It maintains microbial balance by producing bacteriocins and other metabolites, while also suppressing infections and strengthening immunity [2, 3, 5, 6].
It is not associated with virulence or pathogenic factors, making it a Biosafety Level 1 (BSL-1) organism that poses minimal risk 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 indicate a low likelihood of adverse effects for researchers or end-users [1, 9]. L. plantarum is included on the iGEM whitelist [4], which ensures compliance with iGEM safety regulations.
In PRESS, we use these properties to modulate immune responses and reduce airway inflammation associated with asthma [12]. The PRESS project adheres to regulatory probiotic safety standards established by the FDA and EFSA [10, 11].
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 to deposit in the lung region (5×10⁶ CFU) falls within the established safe range for probiotic administration while providing therapeutic benefit. This safe range lies between the least effective dose (10⁶ CFU) and the least lethal dose (10⁷ CFU) [24].
L. plantarum has a strong antagonistic multi-system mechanism against pathogen adhesion and colonization in the respiratory tract [13].
We observed that this multi-system mechanism leads to fewer asthma exacerbations and emergency interventions, while also improving lung function stability by enhancing pathogen resistance and barrier integrity. Additionally, our approach strengthened immune system balance, decreased antibiotic dependency, and improved quality of life, resulting in fewer severe episodes and more stable symptom patterns.
Additionally, our approach strengthened immune system balance, decreased antibiotic dependency, and improved quality of life, resulting in fewer severe episodes and more stable symptom patterns.
This multi-system consists of several mechanisms:
1. Competitive exclusion: Inhibits the adhesion of pathogenic bacteria, including Streptococcus pyogenes, to pharyngeal epithelial cells [14].
2. Barrier function: Reduces the colonization of Streptococcus pneumoniae in lung tissue [15].
3. Receptor competition: Competes for host cell receptors such as angiotensin-converting enzyme 2 (ACE2), potentially preventing viral entry by secreting lipopeptides that bind to ACE2 [16].
4. Direct antimicrobial action: Exhibits antibacterial properties against pathogens such as Pseudomonas aeruginosa and Group A Streptococcus [17].
5. Bacteriocin production: Secretes bacteriocins with antimicrobial effects against Salmonella and Escherichia coli, providing lung protection that extends beyond asthma treatment [18].
We found that L. plantarum can also mitigate pollution effects, including those from particulate matter (PM2.5) and allergens like pollen, which normally cause pro-inflammatory cytokine release (e.g., IL-4, IL-5, IL-13) and IgE antibody production that drive allergic symptoms [19, 20]. Furthermore, it can prevent and treat pollution-induced asthma.
Our research demonstrates that:
1. Lactobacillus 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].
Our findings demonstrate that lipoteichoic acid (LTA) derived from L. plantarum offers immune modulation that is safe and balanced without the harmful inflammatory concerns associated with lipopolysaccharid (LPS)-based treatments. It is perfect for long-term patient treatment since it maintains adjustable dosing with low toxicity while promoting regulatory T-cells and anti-inflammatory responses through TLR2 signaling.
Furthermore, LTA's GRAS certification and straightforward manufacturing procedure provide us with distinct manufacturing and regulatory benefits, establishing it as a workable solution prepared for clinical translation.
| Comparison Aspect | LTA (L. plantarum) | LPS (Gram-negative Vesicles) |
|---|---|---|
| Safety Profile | Low toxicity, well-tolerated at high concentrations, wide therapeutic window | Potent endotoxin with risk of septic shock and cytokine storm, narrow therapeutic window |
| Immunogenic Response | Balanced Th1/Th2 response, promotes regulatory T-cells, anti-inflammatory (IL-10, TGF-β) | Strong Th1 bias, highly pro-inflammatory (TNF-α, IL-1β, IL-6), complement activation |
| Receptor Recognition | TLR2-mediated with CD14 co-receptor | TLR4-MD2-CD14 complex, requires LBP for recognition |
| Clinical Applications | Probiotic therapy, immune modulation, mucosal immunity, chronic inflammatory conditions | Modified vaccine adjuvants, cancer immunotherapy, sepsis research models |
| Long-term Use | Suitable for chronic maintenance therapy, promotes immune homeostasis | Limited to acute interventions, risk of chronic inflammation and endotoxin tolerance |
| Manufacturing | Simple bacterial fermentation, GRAS organism, scalable and cost-effective | Complex vesicle isolation, requires genetic modifications, intricate purification |
| Regulatory Status | GRAS status, established safety profile, Phase II trials, FDA guidance available | Requires extensive safety evaluation, limited approvals, case-by-case review |
| Structure | Polyglycerophosphate chain with D-alanine substitutions and variable lipid anchors | Lipid A core, oligosaccharide chain, O-antigen, hexa-acylated structure |
| Delivery Method | Requires encapsulation for targeted delivery | Natural component of outer membrane vesicles (OMVs) with enhanced cellular uptake |
We found that our bacteria have a natural self-regulatory system called PlnABCD. This system prevents uncontrolled bacterial proliferation and ensures that antimicrobial compound production stays within safe, physiological ranges, preventing disruption of beneficial microbial communities.
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) under liquid culture conditions, as we discovered 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].
In our project "PRESS", we give high priority to lab safety. It is important to predict any accidental problem in a lab experiment and prevent it from happening. So, in PRESS, we expected any problem and found 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.
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. Strict adherence to safety principles throughout our experiments is our top priority. Our laboratory has emergency evacuation route maps, first aid kits, and fire-fighting equipment to ensure preparedness in case of emergencies.
2. All experimental procedures in the lab must strictly follow established norms. Unauthorized operations are prohibited. All equipment requires regular maintenance and must be promptly turned off after use. Reagents must be replaced as needed.
3. Segregation of areas and proper labeling of sample reagents are strictly enforced to maintain organization and safety.
4. Storage of experimental materials is meticulously managed. We store chemicals, instruments, equipment, and experimental tools according to specified guidelines, clearly labeled with details such as name, quantity, and expiration date. We use them responsibly and correctly, clean them after use, and store them in compliance with regulations.
5. Proper waste disposal methods are crucial. Disinfectants must be used to neutralize any remaining bacteria before disposing of waste down the drain. 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.
This year, we have develpoed the this edition of AFCM lab SOPs. It aims to provide information and guidelines to ensure safety and minimize risks when working in laboratories.
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.
Before beginning work in the lab, We were provided with extensive laboratory skills and safety precutions course and pass an examination based on its content.
We were provided with basic laboratory skills and safety precautions during our training and practicing.
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.
Accuracy: when 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.
We found that our weekly lab discussion sessions were invaluable collaborative learning experiences that greatly improved our understanding of complex biochemical concepts. We were good at troubleshooting experimental challenges, exchanging different viewpoints on how to interpret data, and working together to solve problems when our used protocols didn't produce the expected results. We valued how these discussions helped us learn from each other's mistakes and successes. Additionally, the sessions helped us bridge the gap between the theoretical knowledge we learned in the course and the practical application in the lab.
Proper waste disposal methods are crucial. Disinfectants must be used to neutralize any remaining bacteria before disposing of waste down the drain. 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.
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.
Inspired by Edinburgh 2023 team, we developed a reliable secondary containment using olive phenolic extracts that disrupt bacterial cell membranes within minutes while remaining safe and biodegradable within 14-21 days.
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
| 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:
Burns: 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.
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.
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While engineering L. plantarum for our PRESS treatment, we needed additional safety mechanisms that would prevent any unintended risks, either environmental or human health-related, such as leakage to the environment, horizontal gene transfer (HGT), bacterial remnants in the mouth, or translocation across the alveolar-capillary barrier into the bloodstream, causing septicemia. To overcome these issues, we created the PemIK toxin-antitoxin (TA) system to guarantee the safe use of L. plantarum in PRESS.
We decided to develop a toxin-antitoxin (TA) system. Specifically, we chose the PemIK system, as it was 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 bacterial growth by 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].
The PemIK system is a bacterial toxin-antitoxin system found in various bacteria, including L. plantarum [16]. Site-directed mutagenesis confirmed the role of His-59, as either a 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. This acid-base couple is essential for the ribonuclease function, enabling the cleavage of RNA molecules by hydrolyzing the phosphodiester bonds in the RNA backbone [25].
In its native form, although PemI attains conformational stability upon rPemK interaction, it displays vulnerability to proteolysis and is rapidly degraded by the Lon protease, which is a cellular enzyme that targets specific proteins for breakdown [3]. This lability of PemI enables the post-segregational killing (PSK) mechanism, which is called the "addiction module." If the plasmid that expresses the PemIK 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. This creates an 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), known as the addiction module, enhances safety by ensuring that any bacterium that loses its plasmid either via HGT or during replication is 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. This ensures that the desired therapeutic functions and CO-BERA expression are consistently maintained in the same bacterial population, preventing loss of the TSLP-targeting siRNA function and preventing asthma recurrence [4].
The PSK system supports long-term stability by creating a dependency where only plasmid-containing L. plantarum can survive. This reduces the opportunity for HGT to occur in mixed bacterial populations where other species might acquire the plasmid. By using this mechanism, we ensure that the therapeutic dose we determined will remain consistent over time, as CO-BERA will not be expressed by other bacteria in the lung. This prevents reduced immunomodulation effects and maintains optimal CO-BERA expression levels.
To further enhance safety, we integrated the PemIK system with both the phoB promoter and heat-inducible Thermosensor RNA 2U, which collectively form an environmental sensing and response mechanism.
We found that in cases of immunosuppression, bacteria can cross the alveolar-capillary barrier into the bloodstream, and this bacterial translocation causes systemic toxicity, which leads to sepsis. Any asthmatic treatment alternative rather than PRESS has some adverse effects. For example, after corticosteroid inhalation, the remnants in the mouth cause fungal infection, which is known as candidiasis.
We address these conditions through the phoB promoter, which enhances the system's specificity by responding to high phosphate levels that are higher in both the human bloodstream and saliva of the oral cavity than the lungs, ultimately causing bacterial cell death.
The PhoB promoter is activated by the PhoB kinase, which is part of the Pho regulon, in response to phosphate levels:
When phosphate levels are low, PhoR activates PhoB, boosting the expression of genes for both the toxin and antitoxin in our PemIK system.
When phosphate is abundant, PhoR dephosphorylates PhoB, thereby inactivating it and repressing PemIK expression. This leaves PemI to be rapidly degraded without sufficient replenishment, allowing the more stable PemK toxin to dominate and finally leading 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 typically ranging from 3.0 to 4.5 mg/dL [5]. The system is sensitive to phosphate values ranging from 0 to 1000 µM, particularly above 50 µM [28]. Therefore, our phoB promoter will be activated in low-phosphate conditions, 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 reducing the risk of translocation and subsequent sepsis.
In our treatment, given that the average oral cavity temperature is approximately 34°C [30], any bacterial remnants in the mouth will be killed by our engineered toxin-antitoxin system through inactivation of the phoB promoter when high extracellular phosphate levels are present, as the salivary inorganic phosphate level is 1.52±0.63 mmol/l [30]. This prevents oral microbiome disruption, and this feature ensures that our approach promises safer, more effective treatments, paving the way for new medical choices with minimal side effects.
Through our analysis of documented cases, we found that sepsis development typically occurred several days to weeks after initial exposure or risk factors, with highly variable timelines. We identified specific cases showing sepsis development after 9 days, 16 days, and one week across various patient scenarios, including one case where septic shock developed after starting probiotic consumption [31].
Normal serum phosphate levels are found to fall between 2.5 and 4.5 mg/dL. Because infants and children require more phosphate for growth and development, their normal blood levels of phosphate tend to decline with age, reaching their maximum levels in newborns (4.5 to 8.3 mg/dL), which is almost 50% greater than in adults [29].
Additionally, we decided to enhance this TA system by adding a new regulatory component, Thermosensor RNA 2U, to eliminate our L. plantarum if it goes outside its normal niche, such as in cases of laboratory leakage, release through exhalation or coughing, improper handling (e.g., by children), or residues in disposed unsterilized inhalers.
It is known that RNA thermosensors (RNATs) present in non-coding regions of certain mRNAs enable rapid upregulation of protein translation when the temperature of the bacterium rises after entering a mammalian host.
We decided to integrate this system into our bacteria. 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 with increasing temperatures, with the SD sequence occluded at 25°C. 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 heat-inducible RNA-thermosensor 2U, we can confidently say that our system forces L. plantarum to diminish both toxin and antitoxin synthesis under non-physiological and non-intended conditions, allowing the more stable PemK toxin to eliminate the bacteria. Our safety system therefore provides a way to prevent probable risks associated with our L. plantarum bacteria, such as risks related to environmental release, horizontal gene transfer, and unintended survival in the bloodstream, ensuring both patient safety and ecological protection.
Through computational analysis, we found that our bacteria will be killed by our system within 5 hours or even sooner, preventing sepsis, killing both oral and inhaler's remnants that enhances our treatment's safety.
We are pleased to share our approach that preserves both bacterial viability and thermosensor RNA 2U function during and post freeze-drying preparation for our PRESS asthma treatment. We have focused on merging modern technology with reliable safety to ensure L. plantarum delivers its therapeutic effects without any problems.
We developed a comprehensive freeze-drying protocol for our engineered L. plantarum that combines thermal equilibration of heat-inducible RNA thermosensor 2U at 37°C, trehalose vitrification for structural protection, EDTA chelation to prevent both antitoxin degradation and premature toxin activation during processing, followed by controlled EDTA removal and very-low-temperature storage to create a stable dry powder formulation while maintaining bacterial viability, 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. While L. plantarum is recognized as a safe probiotic organism [2], we maintain rigorous safety protocols throughout our project. We use heat-inducible RNA thermosensor 2U that keeps our bacteria confined to the lung environment and prevents horizontal gene transfer (HGT) and any environmental accidents. If these bacteria are exposed to low temperatures, they will definitely be killed. It's critical to ensure that our bacteria stay alive and the heat-inducible RNA thermosensor 2U stays ready to act after freeze-drying.
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Our safety framework integrates multiple protective layers across all project phases, from laboratory containment through patient treatment and environmental protection.
Our framework integrates multiple protective layers. Click a category below to explore the specific risks we've addressed and the mitigation strategies implemented.
Our team is aware that, despite being intended for therapeutic applications, the advanced synthetic biology methods we developed for PRESS may be misused for harmful purposes. We have identified several dual-use concerns that require careful consideration and mitigation strategies.
We acknowledge potential dual-use concerns and have implemented comprehensive measures to prevent misuse while maintaining scientific transparency and therapeutic benefit.
Potential Risk: Our modified L. plantarum could theoretically be repurposed to deliver harmful substances.
Our Solution: We mitigate this by using a GRAS-status, non-pathogenic organism. Crucially, our engineered systems only function under a precise combination of therapeutic conditions (e.g., pH <6.9, specific phosphate levels), making unauthorized use outside this context ineffective.
Potential Risk: The siRNA delivery system (CO-BERA) could be modified to silence essential human genes, causing cellular damage.
Our Solution: Specificity is engineered at multiple levels. We use computational screening (siDirect 2.1) to ensure our siRNA only targets TSLP. Furthermore, our selective packaging mechanism only loads RNA with a specific motif, preventing the inclusion of unintended sequences.
Potential Risk: Our LLO-L461T system could be reverted to its toxic wild-type form to create a cell-damaging agent.
Our Solution: We maintain a 100-fold toxicity reduction through a stable L461T mutation. Additionally, its expression is controlled by a promoter that requires high hydrogen peroxide levels, ensuring it only activates in specific inflammatory environments and remains inert elsewhere.
Potential Risk: The toxin-antitoxin system, designed for safety, ironically involves toxin production mechanisms that could be misapplied.
Our Solution: We selected the PemK toxin specifically because it is a cytosolic endoribonuclease that is only effective against bacteria; it does not harm the human host. The entire system is also dependent on dual environmental sensors, making it non-functional unless very specific conditions are met.
Data Storage: 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 documentation, 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 (phosphate levels, temperature, pH, and H₂O₂ levels) ensure our bacteria only survive and function under very specific conditions. The addiction module creates dependency that prevents survival outside controlled conditions or after plasmid loss, making dual usage difficult. The natural biodegradation of our secondary containment compounds prevents long-term environmental persistence.
Ethical Framework: Our team is dedicated to conducting research ethically, which includes comprehensive risk assessment at every stage of development, expert consultation throughout the process, and transparent reporting of safety measures. We share proven safety methods and contribute to risk assessment frameworks.
Education and Awareness: Our team members undergo training and obtained certification in laboratory safety procedures and dual-use awareness, and we foster a culture where everyone is responsible for detecting and reporting potential misuse. We participate in discussions about the ethical advancement of biotechnology and help develop best practices for the field.
The AFCM-Egypt iGEM team recognized the importance of considering the policy analysis of probiotic-based therapy, so our goal is to examine different aspects of the regulatory frameworks, ethical considerations, market dynamics, and healthcare infrastructure implications related to the advancement, approval, and commercialization of these treatments.
These frameworks play a part in the safety, effectiveness, and quality of probiotic-based medications. Government organizations like the FDA in the US and EDA in Egypt have created clear routes to assess these treatments, which may include quicker approval processes for innovative therapies or regenerative medicines.
Ethical considerations play a crucial role in the creation and use of probiotic-based medications. Crucial issues include patient consent, privacy protection, and fair access to treatments. Ethical guidelines are necessary to ensure informed consent at every stage of the process, from collecting and manipulating probiotics to their therapeutic utilization. Furthermore, it is crucial to ensure fair access to these cutting-edge treatments among different populations and healthcare systems in order to tackle issues of affordability and availability.
Market forces pose major hurdles and possibilities for probiotic-based medications. The significant expenses related to research, development, and production along with uncertainties in reimbursement, affect both the ability to enter the market and the affordability of products. Protecting intellectual property rights is crucial for encouraging innovation and investment in this quickly changing industry. Dealing with these obstacles necessitates strategic measures that promote research and development while also ensuring that healthcare delivery systems are both affordable and sustainable.
The healthcare system needs to adapt to meet the specific needs of probiotic-based treatments. This involves creating sophisticated manufacturing techniques and implementing strong supply chains to guarantee steady production and distribution of probiotic-based products. Incorporating these treatments into current healthcare systems requires training healthcare workers, setting up dedicated treatment facilities, and understanding reimbursement systems to maintain healthcare delivery.
Policy suggestions focused on promoting probiotic-based drugs which involve promoting flexible regulatory strategies that can handle the complex nature of these treatments while still upholding patient safety. Collaborations between the public and private sectors and financial support programs can boost research and development activities. It is essential for regulatory agencies, researchers, and industry stakeholders to work together internationally to align standards, exchange data, and speed up worldwide availability of these game-changing treatments. Providing patients with knowledge and backing advocacy are crucial to guarantee that the advantages, drawbacks, and moral issues of probiotic-based medications are fully acknowledged in policy creation and execution.
In order to ensure the safety of our product development and application, we communicated with EDA (Egyptian Drug Authority) Guidelines and discussed them about drug licensing steps locally and the requirements to comply with regulatory guidelines. Additionally, we asked about the verified clinical trial facilities . These guidelines provide a comprehensive framework for safeguarding public health and the environment by setting standards for research, development, manufacturing, and testing of genetically engineered products. By following these regulations, we able to implement robust safety measures, conduct thorough risk assessments, and maintain transparency throughout the entire process.
To enhance our safety measures, 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 PemIK 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.
Our strategy achieves a balance between safety considerations, biosecurity concerns, and scientific advancement, ensuring that the benefits of our PRESS asthma treatment can be realized while minimizing the potential for harm and misuse. We remain committed to regularly reviewing and improving our dual-use risk management as our project progresses and as new potential applications or concerns emerge.
The PRESS project represents a massive shift in asthma management, moving from symptom-based treatments (e.g., corticosteroids, bronchodilators) to specifically genetically engineered probiotic-based therapies that target 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 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 bacterium, L. plantarum, can be delivered to respiratory tissues by a dry powder inhaler (DPI), offering a non-invasive alternative to systemic drugs with fewer side effects, unlike tezepelumab [3, 4]. While other inhaler-based therapies exist, such as corticosteroids and bronchodilators, our alternative has significantly greater impact. Our therapy lacks the side effects of corticosteroids, which can cause candidiasis, and PRESS has a longer half-life compared to these alternatives, significantly reducing the frequency of required doses.
To prepare PRESS for transition from our laboratory to the clinical field, we conducted successful experiments in the WI-38 cell line. We plan to conduct future preclinical studies in animal models (e.g., ovalbumin-induced asthma in mice) to validate CO-BERA's efficacy in silencing TSLP-mRNA, reducing TSLP-driven inflammation and airway hyper-reactivity [5, 6]. These studies will assess our treatment's delivery efficiency via intranasal administration, biodistribution of L. plantarum, and long-term safety of the TA system in real animal experiments [7]. We plan to collaborate with important regulatory bodies, such as the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA), as this will be essential for PRESS to navigate the approval process for patient therapy.
| Phase | Key Activities | Timeline |
|---|---|---|
| Current (2025) | WI-38 cell line validation, iGEM submission | Complete |
| Preclinical (2026-2027) | Mouse models (OVA-induced asthma), biodistribution | Planned |
| IND Enabling (2027-2028) | GLP toxicology, manufacturing scale-up | Future |
| Clinical Phase I (2029+) | Safety in healthy volunteers | Target |
| Clinical Phase II/III | Efficacy in asthma patients | Long-term |
Beyond asthma, the design of PRESS, which uses dual-promoter regulation (pKatA and p170-CP25), can be used to target other inflammatory or infectious diseases with the same pathogenic mediator, TSLP, and the same environmental conditions of pH and hydrogen peroxide. For instance, CO-BERA could be re-engineered to target other mRNAs associated with COPD or viral infections (e.g., SARS-CoV-2 spike protein) [8, 9].
Our TA system's environmental sensors (Heat-inducible RNA-thermosensor 2U and PhoB promoter) could be adapted to sense other cues, such as oxygen levels or microbial metabolites, to expand applications for treating gut-related disorders like inflammatory bowel disease [10, 11].
To build trust and acceptance for our therapy and decrease GMO stigma, we engaged stakeholders, patients, and healthcare providers through workshops and webinars. These efforts addressed ethical concerns about GMOs and specifically about our therapy, including potential misuse of TA systems for bioterrorism [12].
Genetically modifying cells for therapeutic purposes raise ethical concerns. Informed consent, potential risks versus benefits, and long-term consequences for patients need careful consideration and open communication.
We conducted a survey with bronchial asthma patients along with doctors (pulmonologists and allergists) in addition to online surveys but before that they signed informed consent implying that they accepted entering the PRESS project survey on L. plantarum treatment via DPI voluntarily.
The informed consent is found below:
To reduce negative environmental effects and guarantee the long-term sustainability of probiotic-based treatments, the PRESS project prioritizes sustainability. In line with the United Nations Sustainable Development Goals (SDGs), our strategy incorporates ecological, economic, and social aspects.
We hope to transform respiratory disease management while minimizing ecological and social risks by integrating effective biocontainment (PemIK system, Heat-inducible RNA-thermosensor 2U, 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 facilitating progress for other iGEM teams. The long-term vision of our PRESS project is to establish a safe, effective, and scalable platform for microbial therapeutics, with asthma as a proof-of-concept.
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