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.
Probiotic Inhibition of Pathogen Adhesion and Binding
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].
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.
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