Overview

iGEM has always urged each team to abide by Safety & Security, and has made strict requirements in this regard. Safety focuses on the identification and control of "unexpected incidents" risks. iGEM requires teams to demonstrate how experiments and designs can minimize potential harm to humans, animals, and the environment. Security focuses on the risk of "being abused or misused by humans". iGEM requires teams to identify possible dual-use risks and take mitigation measures.
Guided by the spirit of iGEM, our team promotes innovation and encourages initiative, and working safely and reliably is the prerequisite for active innovation. We highly value the principle of "being a responsible scientist or engineer", especially prioritizing the avoidance of harm to ourselves, colleagues, and the environment. Therefore, safety and security permeate our project.
We keep in mind that "being transparent about possible risks and how we manage these risks is a key component of being a responsible scientist or engineer". Therefore, we have identified potential risks in advance and proposed relevant countermeasures in experiments and future product applications. It is worth noting that our safety concept runs through the entire project. Not only does it consider the unique safety of conducting project-related experiments, but also conducts safety design based on the entire product line sequence. Moreover, we adhere to the highest standards of laboratory safety, promote the importance of safety in human practice, ask experts how to conduct safety risk management, and abide by relevant laws and regulations.
Here is our self-inspection form regarding our work.

Here, we also present the self-check form regarding the iGEM Safety Policy and its derived rules:

Safety

Laboratory Safety

1. General laboratory safety

2. Personal safety protection

3. Experimental safety considerations for the uniqueness of the project

Reference

1. Laboratory Safety

1.1 General laboratory safety

(1) laboratory equipment

The project experiments were conducted in two core laboratories of the Naval Medical University: the high-performance liquid chromatography (HPLC), immunofluorescence, and microplate reader analyses were carried out in the central laboratory, while all other experiments were completed in the iGEM laboratory of the Department of Biophysics.
All the experimental operations involving live Genetically Modified Microorganisms (GMMs) in the project were carried out in Biosafety Level 2 ( BSL-2) laboratories. This choice provided a solid and reliable physical guarantee for all experimental activities. The layout and facility configuration were carefully planned to maximize the safety of laboratory personnel, research materials, and the external environment. The orderly layout of the laboratory ensured the efficiency and safety of the workflow (Photo 1).
The core hardware facilities of the laboratory (Photo 2) form the material basis of its safety protection system:

a. Flame-retardant and waterproof workbench: The workbench surface is made of flame-retardant and waterproof materials, reducing the risk of fire or chemical hazards caused by accidental splashing or high-temperature operations.

b. Mechanical ventilation system: The laboratory is equipped with a mechanical ventilation system, and the exhaust system is specially equipped with filters, aiming to effectively filter the possible aerosols and prevent microorganisms or harmful substances from escaping into the external environment, thereby maintaining the air cleanliness of the laboratory and the safety of the external environment.

c. Class II biological safety cabinet (BSC): The key engineering control measure is the use of Class II biological safety cabinets. All operations involving the transfer, cultivation, transformation, and analysis of live bacteria, etc., are completed in the biological safety cabinets. The biological safety cabinet serves as a primary physical barrier, protecting the laboratory personnel through directed airflow and protecting the experimental materials and environment through filtered air, and is the core equipment to ensure the safety of microbial operations.

d. Chemical fume hood: The chemical fume hood is the key safety barrier of the laboratory. Its main function is to effectively isolate and exhaust toxic, corrosive, or flammable chemical gases and aerosols produced during the experiment through a negative pressure exhaust system, protecting the operators from inhalation exposure hazards and preventing the accumulation of chemical vapors indoors, ensuring the safety of the experimental environment.

e. Emergency and sterilization equipment: The laboratory is equipped with a high-pressure sterilizer and other sterilization equipment. The water pipes are equipped with backflow preventers. In addition, there are showers, eyewash stations, fire-fighting equipment, first aid equipment, and emergency lighting devices, forming a complete emergency response system to ensure that effective measures can be taken promptly in the event of an emergency.

Fig 1. Our laboratory

Fig 2. Our equipment

(2) Waste management and disposal solution: A closed-loop system ensuring safe circulation

To prevent potential hazards to the environment and personnel caused by laboratory waste, the project team has established a systematic and institutionalized waste disposal closed-loop system. This system ensures that the entire process of waste generation to final disposal is strictly controlled.

a. Solid biological hazard waste disposal: All solid wastes that have come into contact with GMMs, including petri dishes, pipette tips, centrifuge tubes, and gloves, must be collected in a dedicated, yellow garbage bag with a biohazard label. Before final disposal, these wastes must be thoroughly inactivated by autoclaving (121°C, pressure 15psi, for at least 20 minutes) to ensure that all microorganisms are completely killed. After inactivation, the waste can be treated as ordinary medical waste. All sharp items (such as needles, blades) must be discarded separately in a puncture-resistant, dedicated sharps container.

b. Liquid biological hazard waste disposal: Contaminated liquid media and buffers containing GMMs must not be directly discharged into the sewer. The preferred method is autoclaving. An alternative method is chemical disinfection, where a solution of effective chlorine concentration of at least 10% (hypochlorous acid solution) is added to the waste liquid, and the contact time must be no less than 30 minutes. All waste liquid will be finally poured into designated collection buckets and uniformly handled by the institution's professional department.

c. Chemical waste disposal: During the in vitro PET degradation experiment, the waste liquid containing degradation products such as TPA, MHET, and BHET generated will be treated as chemical waste. Before disposal, the team will consult the material safety data sheet (MSDS) of the relevant chemicals and strictly follow the institution's regulations on chemical waste disposal. The waste will be collected in a dedicated chemical waste bucket and handed over to the school's environmental health and safety (EHS) department for unified recycling and disposal.

The following table provides a clear overview of the disposal plans for various types of waste in the laboratory:

(3) Safety operation principles and emergency response mechanism: Comprehensive coverage from prevention to control

The project team has established a multi-level and comprehensive set of safety operation principles and emergency response mechanisms to ensure that every aspect, from daily prevention to emergency control, is effectively managed.
a. Daily operation norms and personnel behavior guidelines
a.1 Access control: The laboratory implements a strict access registration system to ensure personnel tracking and safety management.
a.2 Personal protective equipment (PPE): During the experiment, the mandatory use of goggles, protective gloves, laboratory coats, isolation suits, etc. is required to maximize protection for the laboratory personnel.
a.3 Behavioral norms: Eating and littering are strictly prohibited within the laboratory to ensure a clean and safe operating environment.
b. Leak accident handling plan
The laboratory is equipped with an emergency leak handling kit, including adsorption materials, forceps, 70% ethanol, and 10% bleach solution as disinfectants. In case of GMM leakage, the following steps should be immediately executed:
b.1 Immediate action: Immediately cover the contaminated area with adsorption materials and soak it with sufficient 10% bleach solution.
b.2 Inactivation treatment: Ensure that the disinfectant acts for at least 30 minutes to completely inactivate the microorganisms.
b.3 Waste disposal: All the waste generated during the cleanup process, including adsorption materials and gloves, must be treated as biological hazardous materials.
b.4 Leakage in the biosafety cabinet: If the leakage occurs inside the biosafety cabinet, all items and surfaces within the cabinet should be thoroughly disinfected while maintaining the operation of the safety cabinet.
c. Unexpected exposure handling plan
For different types of unexpected exposures, clear response measures have been formulated:
c.1 Skin or eye contact: If the skin accidentally comes into contact with the bacterial solution, immediately rinse with soap and flowing water for at least 15 minutes. If the eyes are splashed with the bacterial solution, immediately go to the eyewash station and rinse the eyes with mild flowing water for at least 15 minutes.
c.2 Systemic exposure: For more serious incidents, such as needle punctures, cuts, ingestion, or inhalation, the laboratory supervisor and the school's biosafety office must be reported immediately, and professional medical assessment must be sought.
d. Environmental release emergency plan
Although the inherent biological protection system designed by the project team (i.e., GMMs lack specific survival conditions and cannot survive in the natural environment) significantly reduces the possibility of environmental release, a detailed emergency plan has still been formulated. The primary principle is "control and inactivation". Specific steps include:
d.1 Area lockdown and disinfection: Immediately seal off the contaminated area and use appropriate disinfectants (such as bleach powder or peroxide acetate solution) to thoroughly disinfect the leakage material and the surrounding environment.
d.2 Reporting process: Immediately report to the institution and necessary national regulatory authorities. The report will detail the identity of the leaked microorganisms (strain, genetic modification details), the volume and concentration of the leakage, the location, and the control measures already taken.

Fig 3. Our exit route and firefighter kit

Fig 4. Our general risk management

1.2 Personal safety protection

Before experimenting, our PI provided us with comprehensive laboratory training. The training content was very extensive and was passed through both teaching and testing, including basic experimental skills, laboratory cleaning, waste disposal, ethical guidelines, chemical storage, laboratory rules, equipment safety, personal protection, and emergency plans. Additionally, during our experiment, at least one experienced teacher was present to provide necessary guidance and ensure the safe conduct of the experiment.

Fig 5. The instruction from our PI

(1) Experimental skills training
Basic laboratory skills training covers a range of specific techniques, including plasmid extraction, plasmid transformation, PCR, bacterial inoculation and amplification, cell lysis, liquid chromatography, agarose gel electrophoresis, spectrophotometer operation, and laser confocal microscopy. Laboratory safety precautions encompass not only meticulous documentation of instrument usage and accurate sample labeling, but also strict adherence to waste disposal protocols. ( Standard operating procedures for instrument use and waste disposal ) In the event of an accident, the instructor will demonstrate emergency evacuation routes and teach how to safely utilize the laboratory's first-aid kit, firefighting equipment, and relevant emergency response procedures. (Emergency Plan for Laboratory Accidents )

To address the risks of Saccharomyces cerevisiae and microplastics, the PI provided us with detailed instructions. Personnel who have not received safety training or have not demonstrated proficiency in basic laboratory skills are not allowed to participate in the experiments. This approach ensures the acquisition of more rigorous, accurate, and reliable experimental data. By implementing these training measures, we have maximized the safety and stability of the experiments, thereby reducing potential risks to the environment and ourselves. During the experiments, we have always maintained a rigorous and conscientious attitude, striving to ensure that every detail meets the standards. Through continuous efforts, we believe that laboratory work will become more efficient, safe, and reliable.

(2) Personal protection training
In addition to laboratory skills training, each member actively engaged in learning about personal protection measures and strictly followed laboratory procedures to reduce potential risks as much as possible (Photo 4). The overview of personal protection training is as follows:
Dressing: In the laboratory environment, the laboratory coat should cover the knees and the sleeves should be wrapped around the wrists to protect the skin and personal clothing, avoiding direct contact with harmful substances. It is essential to wear the standard long-sleeved laboratory coat, long pants, gloves, and a mask to ensure that your hair is properly restrained. Jewelry that may affect the safety of the experiment is strictly prohibited. Shoes should be fully covered, and no shoes with heels or slippers should be chosen.
Cleaning:Before entering and leaving the laboratory, one must thoroughly wash hands. If there is obvious hand contamination when removing gloves, wash your hands first before continuing to remove other personal protective equipment. Use soap and water or alcohol for washing. In addition to daily cleaning and disinfection measures, the performance of the clean workbench needs to be tested and evaluated regularly.
Equipment Usage:
a. Before use, carefully check if all conditions meet the requirements and if there are any abnormalities; during use, pay attention to observing whether the various states of the instrument are normal and make adjustments if necessary; after use, complete, clean, or blow off and turn off the instrument.
b. Before using any machine, receive appropriate training. All equipment use must be registered and strictly follow the instructions. Before the experiment, check the equipment to ensure it is running normally and prevent equipment failure. During operation, the condition of the equipment should be monitored at any time to detect any abnormal situations.
c. Equipment maintenance is divided into regular maintenance and daily maintenance. The purpose is to identify potential faults so that preventive measures can be taken in time. Regular maintenance is the fixed-time maintenance of complete large equipment. Daily maintenance is the daily maintenance and inspection of the equipment, including spot checks per shift and daily inspections.
d. After the experiment, thoroughly clean the equipment and check to prevent the presence of residual harmful substances and bacteria.
Reagent usage: To obtain accurate experimental results, the following rules should be followed when using the reagents to ensure they are not contaminated and do not deteriorate:
a. Do not touch the reagents with your hands.
b. Use clean spoons, graduated cylinders, or droppers to take the reagents and never reuse the same tool to take multiple reagents.
c. After using the reagents, the cork must be tightly closed. The cap and dropper should not be placed in the wrong position, and the bottle should be returned to its original position.
d. The taken reagents should not be put back into the original reagent bottle.

Fig 6. Laboratory safety regulations and partial emergency tools

1.3 Experimental safety considerations for the uniqueness of the project

(1) Regarding the chassis bacteria - Saccharomyces cerevisiae

Safety of the experiment on the Saccharomyces cerevisiae
Saccharomyces cerevisiae, as a low-risk chassis organism widely used in brewing, baking, and industrial fermentation, is highly favored in the field of synthetic biology. Its safety has been recognized by multiple authoritative institutions. The U.S. Food and Drug Administration (FDA) has classified its extract as "Generally Recognized as Safe" (GRAS), and the U.S. National Institutes of Health (NIH) has listed it as a safe organism in its "Guidelines for Research Involving Recombinant DNA Molecules". In iGEM and conventional laboratory settings, Saccharomyces cerevisiae is classified as a BSL-1 risk group 1organism and has been included in the iGEM white list, posing minimal potential harm to healthy adults, the environment, and the public. The risk of infection is extremely low, and even if an infection occurs, it is usually not dangerous and is easy to treat. Although Saccharomyces cerevisiae itself has low-risk characteristics, this does not mean that laboratory operations are completely risk-free. A comprehensive assessment of the risks associated with Saccharomyces cerevisiae requires going beyond its inherent biological attributes and conducting an in-depth analysis of the "secondary" risks introduced by experimental operations or specific genetic modifications.
a. Fungal contamination risk: Many common molds can grow well on yeast culture media and compete effectively, potentially covering the yeast colonies. Therefore, the focus of contamination control in yeast experiments may lie in preventing fungal contamination, while bacterial contamination is relatively easier to distinguish.
b. Aerosol transmission: The release of microbial aerosols in the laboratory is an important source of infection risk, potentially affecting all laboratory users. Many laboratory procedures have the potential to generate aerosols, such as centrifugation, pipetting, etc. For fungi, especially those that form spores, aerosols are one of the main transmission routes of fungi in the laboratory. Therefore, when performing centrifugation or other operations that may generate aerosols involving yeast, special attention must be paid. To prevent aerosol transmission, operations must be carried out inside a biosafety cabinet and use leak-proof secondary containers or sealed rotors with gaskets.

Culture, transformation, and gene editing of Saccharomyces cerevisiae
a. Adhere to general laboratory safety and personal safety protection principles
b. Utilize special nutritional defect screening to reduce strain leakage
Mechanism principle: Nutritional defect (Auxotrophy) is achieved through gene knockout technology, by deleting a key gene in the pathway where microorganisms synthesize a certain essential metabolite (such as amino acids, vitamins), making them completely dependent on the external environment for the supply of this metabolite to survive. This is a widely verified and genetically extremely stable biological protection method.
Design implementation: Saccharomyces cerevisiae: We precisely knock out the his and/or ura genes in its genome. These genes encode enzymes that are an indispensable part of the histidine and uracil biosynthesis pathway. Therefore, the modified yeast strain will become a histidine and/or uracil nutritional defect type, and can only grow when additional histidine and/or uracil are added to the culture medium.

a. Safety principles for Saccharomyces cerevisiae cultivation: Saccharomyces cerevisiae Cultivation Safety PDF (see file)

b. Safety precautions for yeast transformation experiments: Saccharomyces cerevisiae Transformation Safety PDF (see file)

c. Safety precautions for yeast genome editing experiments: Saccharomyces cerevisiae Genome Editing Safety PDF (see file)

Coating of Saccharomyces cerevisiae
The safety precautions for the sodium alginate-chitosan microcapsule coating experiment are as follows:

Table 1. The safety of coating steps

In response to these safety issues related to yeast, we have employed a variety of methods in the laboratory to implement the safety requirements of iGEM. The more common ones include: rigorous experiments in a laminar flow hood, screening for nutritional deficiencies, and microcapsule encapsulation etc.

(2) Safety precautions for Saccharomyces cerevisiae spores

After Saccharomyces cerevisiae was included in the whitelist, we reminded the team to be aware of the threat posed by its spores. We carefully considered this issue.
Yeast spores are dormant carriers of genetic material and have the following risks:
Persistence in the environment: The spores have strong resistance to dryness, ultraviolet rays, and common disinfectants and can survive outside the laboratory.
Transmissibility: The spores are small in size and light in weight, and are extremely easy to spread through air currents, personnel operations, or equipment surfaces, causing cross-contamination or environmental release.
Genetic risk: The spores can lead to genetic material exchange through sexual reproduction. If the engineered strain spores escape, it may bring about unpredictable ecological impacts.
Laboratory measures

Following a unique spore processing procedure, See “Spore Laboratory Processing Documents”

Engineering measures
Mechanism principle: Spore formation elimination is achieved by targeting and knocking out the core regulatory genes involved in meiosis and spore formation processes, thereby causing the yeast to completely lose its ability to produce spores. This strategy is highly genetically stable and specific, and is a powerful biological protection method for preventing spore contamination at its source.
Design and implementation: In the diploid strain of Saccharomyces cerevisiae, we precisely knocked out the IME1 gene. This gene encodes the transcriptional activator Ime1p, which acts as a core integrator responding to nutritional signals (lack of glucose/nitrogen sources, presence of non-fermentable carbon sources) and cell types (specific to the MATa/MATα diploid), and is necessary for initiating the meiosis and spore formation programs. The ime1 knockout strain can still proliferate normally through mitosis and maintain metabolic functions, but is completely unable to enter meiosis and form spores. Studies have shown that the ime1 knockout wine yeast maintains its original biotechnological performance during fermentation and has no obvious negative effects^[1].
Phenotypic verification: The engineered strain and the wild-type strain were co-inoculated into the spore formation induction medium (such as acetic acid potassium medium), and cultured under suitable conditions with continuous microscopic observation. The wild-type strain should form typical tetrad spore structures, while the ime1 knockout strain shows no spore formation at all, proving that its spore formation ability has been successfully eliminated.

(3) Regarding the degradation target - microplastics

Basic Concepts of Risk
Microplastics pose several laboratory risks:
Inhalation: Fine particles (smaller than 10 µm, especially smaller than 2.5 µm) may reach deep lungs and cause inflammation or cellular damage.
Ingestion: Indirect intake through contaminated hands, food, or beverages.
Eye contact: Possible mechanical irritation.
Environmental contamination: Easily carried outside via air, clothing, or tools.
Static electricity: Causes particles to disperse and adhere to surfaces.

Engineering and Control Measures
Storage: Keep powders in sealed, clearly labeled containers (Eg,' Microplastic—Avoid inhalation'). Store in a dry, stable cabinet.
Operation:
• Perform weighing, transferring, and suspension preparation only in a fume hood or biosafety cabinet.
• Use antistatic weighing materials and gentle handling.
• Prefer wet operation to minimize dust.
• Conduct vortexing or centrifugation in closed containers.

Personal Protective Equipment (PPE)
Respiratory: Wear N95/KN95/FFP2 masks (PAPR for high concentrations).
Eye: Use dust or chemical goggles.
Hands: Nitrile gloves; remove carefully inside the hood.
Body: Laboratory coats with tight cuffs; do not wear outside the lab.

Waste Disposal
Solid waste: Place gloves, papers, and tubes in sealed, labeled containers for authorized disposal.
Liquid waste: Collect separately; do not pour into drains.
Cleaning materials: Treat all contaminated wipes or adsorbents as microplastic waste.

Emergency Response
Alert personnel and isolate the area.
Wear PPE before cleanup.
Do not sweep or vacuum. Use wet towels or absorbents to collect spilled material.
Collect all waste into sealed, labeled containers.
Wipe the area repeatedly with water and a cleaning agent; collect wastewater.
Report incidents and seek medical care if exposed.

Management and Training
Establish a Microplastics SOP and ensure all staff follow it.
Provide safety training covering PPE use, spill handling, and waste management.
Keep records of microplastic use and disposal to match actual inventory.

(4) Regarding gene editing technology - CRISPR/Cas9

Concept and Application Overview of “Safe Design

In this project, the use of CRISPR/Cas9 technology follows the "Safe-by-Design" concept, aiming to actively eliminate potential biological safety risks.
Core Project Applications
Targeted knockout of certain genes in the chassis organism - Saccharomyces cerevisiae. Mainly includes:
Gal80: Used to construct a glucose-related dynamic regulation system to control the function of the adsorption module, enabling more efficient expression.
Sec72: Knockout can enhance the expression of NCW2, significantly increasing the secretion efficiency.
IME1: A key gene for spore formation, knocking out it makes Saccharomyces cerevisiae unable to produce spores.
Integration of the target gene into the Saccharomyces cerevisiae genome. Mainly includes:
a. MHETase: In the experiment, we failed to transform MHETase, so we chose to integrate this enzyme into the genome. Research shows that this can also reduce metabolic toxicity (A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly).
b. Therapeutic fusion gene
c. Construction of HSP26-EGFP when verifying the HSP26 promoter

Core Security Statement: No gene drive

This project hereby declares that no form of gene drive has been designed, constructed, or tested.
The project design strictly avoids any technical elements defined by the iGEM gene drive policy.
Transient expression and non-integration: The Cas9 nucleases and guide RNA (gRNA) are expressed transiently through non-integrating, non-self-replicating plasmids. The editing tools will be lost along with cell division after the task is completed and will not integrate into the host genome.
Lack of core driving mechanism: The core feature of gene drive is to integrate the gene encoding the nucleases into the host genome, enabling it to self-replicate and super-mendelian inheritance. In this project, the Cas9 gene is always located on a plasmid outside the chromosome and does not have a driving effect.
Strict containment: All operations are conducted in compliant biosafety laboratories and strictly abide by the "prohibition of environmental release" policy of iGEM.

Comprehensive risk assessment and control for biosafety

All CRISPR/Cas9 operations are conducted under a comprehensive biosafety and biosecurity framework. Experiments take place in institution-approved BSL-2 laboratories equipped with biosafety cabinets to prevent aerosol dispersion. All participating members have completed certified BSL-2 and recombinant DNA safety training, with detailed SOPs established for transformation, screening, and waste inactivation to minimize human error.
At the design level, safety is ensured through strict host and vector control. The gene editing host is the laboratory strain Saccharomyces cerevisiae, listed as a Risk Group 1 organism on the iGEM whitelist. The gRNA targets the nonessential IME1 gene, related only to sporulation and confirmed bioinformatically to have no homology with virulence factors or allergens. The plasmid used is non-conjugative and lacks horizontal gene transfer elements, preventing spread to other microorganisms. Nutritional markers (e.g., URA3) are used instead of antibiotic resistance genes.
Functionally, off-target risk is controlled through predictive bioinformatics screening and sequencing-based verification. The engineered strain includes both passive and active containment mechanisms: nutritional deficiencies prevent survival outside specific media, and an inducible kill switch ensures self-elimination under defined conditions.
Biosecurity measures are implemented to prevent dual-use risks. Sensitive gRNA sequences are withheld from public release, while all DNA fragments undergo sequence screening against pathogen and toxin databases. Only authorized personnel can access CRISPR materials and data, and all experimental procedures are logged via electronic laboratory notebooks to ensure traceability and accountability.
This multi-layered system ensures that CRISPR/Cas9 use in this project remains safe, contained, and compliant with iGEM and institutional biosafety standards.

Compliance and evidence index

This project strictly adheres to official iGEM policies and institutional and national regulations. All required safety forms have been submitted as mandated and approved by the Institutional Biosafety Committee (IBC). All integrated genes comply with safety standards, and the genomic targets employed will not affect the normal functions of Saccharomyces cerevisiae. Target information is provided in the attachment.

Furthermore, before the experiment, we conducted some thinking based on the above content and created a checklist for using CRISPR/Cas9, which was continuously updated. This checklist covers the key safety and security considerations required when using CRISPR/Cas9 in the iGEM project. It particularly emphasizes isolation measures, non-targeted analysis, exclusion of gene drive applications, risk assessment, and the safe handling of biological and digital resources.
The main content is as follows. For the detailed checklist, please refer to theattachment.

(5) Other specific safety precautions for the experiment

a. Safety precautions for HPLC experiments
a.1 Chemical safety related to HPLC experiments
1. Acetonitrile (MeCN) is toxic, volatile, and flammable. It must be handled in a fume hood.
2. Formic acid and buffers may irritate the skin and eyes. Therefore, they should be handled with care.
3. Standard samples of TPA, MHET, and BHET should be prepared in small quantities, stored in individual bottles, and labeled properly.
a.2 Instrument operation related to HPLC experiments
1. HPLC is to be operated only by trained personnel.
2. Pay attention to the pressure cap, dismantling the pipeline under high pressure in the system state.
3. The boot operation is performed before checking whether the mobile phase, sample bottles, and waste liquid bottle are placed correctly.
a.3 Waste liquid and consumable treatment related to HPLC experiments
1. All waste liquids containing acetonitrile/methanoic acid/buffer solutions must be centrally collected in clearly labeled waste containers and handled uniformly by the laboratory.
2. Discarded filter membranes, injection needles, and other consumables should be placed in the chemical waste bin and must not be thrown into the ordinary trash can.

Fig 7. High-Performance Liquid Chromatography

b. Safety precautions for electron microscopy experiments
b.1 Chemical Safety related to electron microscopy experiments
1. Glutaraldehyde (2.5%): Highly irritating and toxic. It should be handled in a fume hood, avoiding inhalation of vapors or skin contact.
2. Osmium acid (OsO₄, 1%): Extremely strong oxidizing property and high toxicity. It must be handled in a fume hood, wearing double-layer gloves and a protective face mask.
3. All waste liquids should be immediately collected into the dedicated waste liquid bottle for osmium acid and must not be discarded randomly.
4. Organic solvents (ethanol, acetone): Flammable. Keep away from fire sources. Handle in a fume hood and ensure the waste liquid enters the organic waste liquid bucket.
5. Uranium staining agent, lead citrate: Low radioactive risk (commonly used as weak radioactive isotopes or substitutes for salts), but with heavy metal toxicity. Gloves must be worn during the operation, and the waste liquid should be concentrated and collected into the heavy metal waste liquid bucket.
b.2 Instrument Safety related to electron microscope experiments
1. The operation of the TEM instrument is restricted to personnel who have received training.
2. The operation of the microtome (ultra-thin sectioning machine) involves a risk of mechanical cuts. It must be operated by trained personnel, with hands kept away from the blade.
3. The sample mesh of the TEM needs to be handled gently to avoid bending or contamination.
b.3 Handling of waste liquids and consumables related to electron microscopy experiments (Waste Disposal)
1. Classification of chemical waste:
Glutaraldehyde, osmium acid → Highly toxic waste liquid bottle.
Ethanol, acetone → Organic solvent waste liquid bottle.
Uranium, lead staining solution → Heavy metal waste liquid bucket.
2. Solid waste (used filter paper, gloves, plastic consumables) should be collected separately, labeled, and handed over to the laboratory safety officer for unified disposal.

c. Safety precautions for immunofluorescence experiments
c.1 Chemical safety related to immunofluorescence experiments
1. Paraformaldehyde (PFA): It is highly toxic and irritating, and has been confirmed as a carcinogen. All operations involving PFA solids or solutions must be conducted in a chemical fume hood.
2. Triton X-100: It can cause severe eye damage and skin irritation. During operation, chemical safety goggles must be worn, and ensure that the eyewash station is available.
3. Mounting agents and slide coating agents: Please refer to their safety data sheets (SDS) before use. Some mounting agents are flammable, and coating agents such as ConA (Concanavalin A) may be allergens.
c.2 Instrument operation related to immunofluorescence experiments
1. Centrifuge: The centrifuge tubes must be precisely balanced by weighing (instead of visually estimating the volume). Before the operation, check for any cracks in the centrifuge tubes and the rotor, and ensure that the lid is opened only after the rotor has completely stopped.
2. Confocal Laser Scanning Microscope (CLSM): It contains high-power lasers and poses a serious risk to the eyes. It is only for operation by trained personnel. It is strictly prohibited to disassemble any protective enclosures or bypass the safety interlock devices.
3. General Specifications: Throughout the experiment, one must wear laboratory coats, nitrile gloves, and safety glasses. In steps that may cause splashes (such as handling PFA, Triton X-100), higher-grade chemical goggles with protection should be worn.
c.3 Handling of waste liquids and consumables related to immunofluorescence experiments
1. Chemical waste liquid: All waste liquids containing polyethylene glycol and Triton X-100 must be collected in clearly labeled hazardous chemical waste containers and handled uniformly by professional institutions.
2. Chemical solid waste: All consumables contaminated by hazardous chemicals such as PFA (polyfluoroalkyl substances) (e.g., pipette tips, gloves, centrifuge tubes) must be discarded in designated chemical solid waste containers.
3. Sharps: Used or broken slides and coverslips must be disposed of in dedicated sharps containers.

Fig 8. Laser Confocal Microscope

d. Safety precautions for the use of microplate readers
i. Biological and chemical safety related to microplate reader experiments
1. Biosafety: The Saccharomyces cerevisiae used in the experiment is a BSL-1 microorganism and does not cause disease in healthy adults. The operation can be carried out following standard microbiological procedures.
2. Aseptic operation: Strain inoculation and 96-well plate addition should be performed in a laminar flow hood or biosafety cabinet to prevent sample contamination and aerosol generation. The work surface should be disinfected before the operation.
3. Personal hygiene: Eating, drinking, or applying makeup is strictly prohibited within the experimental area. After handling biological samples, after removing gloves, and before leaving the laboratory, hands must be thoroughly washed.
4. Fire prevention: If using an alcohol lamp or Bunsen burner for aseptic operation, ensure there are no flammable substances such as alcohol nearby, and always pay attention to the safety of the flame.
ii.Instrument operation related to microplate reader experiments
1. Enzyme reader operation: Only trained personnel are allowed to operate it. Before running, make sure the 96-well plate is securely placed in the plate holder to prevent movement or spillage during high-speed oscillation.
2. High-pressure sterilizer: The sterilization of culture media involves high temperature and high pressure. It must be operated by personnel who have received specialized training and strictly follow the equipment safety procedures.
iii. Waste liquid and consumables treatment related to microplate reader experiments
1. Biological waste liquid: Liquid wastes such as yeast culture solutions must undergo chemical inactivation (e.g., adding bleach to a final concentration of 10% and leaving it for 30 minutes) or high-pressure steam sterilization before being discharged into the sewer.
2. Biological solid waste: All consumables that have come into contact with bacterial solutions, such as 96-well plates, pipette tips, centrifuge tubes, etc., must be collected in dedicated biological hazard waste bags and undergo high-pressure steam sterilization before being discarded as ordinary garbage.
3. Work area purification: After the experiment, all used work surfaces must be cleaned with effective disinfectants such as 70% ethanol or 10% bleach.

Fig 9. Enzyme-Linked Immunosorbent Assay Reader

References

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8.Ponomarova, O. et al. Yeast creates a niche for symbiotic lactic acid bacteria through nitrogen overflow. Cell Syst. 5, 345-357.e6 (2017).
9.Nenciarini, S. et al. Impact of cooperative or competitive dynamics between the yeast saccharomyces cerevisiae and lactobacilli on the immune response of the host. Front. Immunol. 15, 1399842 (2024).
10.Characterization of extracellular yeast peptide factors and their stress-protective effect on probiotic lactic acid bacteria - PubMed. https://pubmed.ncbi.nlm.nih.gov/28853771/.
11.He, X., Liu, B., Xu, Y., Chen, Z. & Li, H. Effects of lactobacillus plantarum on the ethanol tolerance of saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 105, 2597–2611 (2021).
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14.U.S. Food and Drug Administration. (2024). CFR - Code of Federal Regulations Title 21, Part 184, Sec. 184.1724: Sodium alginate.
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17.https://2022.IGEM.wiki/athens/safety
18.https://2021.IGEM.org/Team:SZU-China/Design
19.https://2023.IGEM.wiki/ptsh-taiwan/kill-switch
20.https://2022.IGEM.wiki/gems-taiwan/experiments
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22.https://2020.IGEM.org/Team:Fudan/Design/kill_switch
23.https://2021.IGEM.org/Team:Vilnius-Lithuania/Contribution）
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27.https://2023.IGEM.wiki/ptsh-taiwan/kill-switch
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29.https://2020.IGEM.org/Team:Calgary/Biocontainment_Engineering
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36.Zhang W, Jiang X, Yang Y, et al. L-arabinose exerts probiotic functions by improving gut microbiota and metabolism in vivo and in vitro. Food Funct. 2022;13(8):4678-4690. doi:10.1039/D2FO00161B

Safety

The problem of metabolic toxicity

1. Modeling - FBA Analysis

2. HP exploration

3. Experimental and circuit design measures

Product safety

1. Product form

2. The use of the product

Part and Material Safety

1. Overview

2. Parts

3. Materials

1. The problem of metabolic toxicity

1.1 Modeling - FBA Analysis

Throughout the design and implementation of our project, safety has remained our highest priority. Beyond standard biosafety risk assessments, we focused on two categories of potential risks that are intrinsically linked to our system design:

a. Metabolic Toxicity Analysis — Within the framework of the iGEM Safety Guidelines (2023), metabolic toxicity analysis serves as a critical bridge between system feasibility and biological safety. The introduction of heterologous gene circuits can alter the metabolic flux distribution of the chassis cell, leading to competition for energy resources, precursor depletion, and accumulation of metabolic intermediates—collectively resulting in metabolic stress and potential cytotoxicity. Such imbalances may not only suppress host growth but also trigger mutations or metabolic drift, posing risks to system stability and ecological safety. To ensure that our engineered strain can maintain normal growth while expressing exogenous proteins, we employed Flux Balance Analysis (FBA) to quantitatively evaluate metabolic toxicity, confirming the stable performance of our engineered yeast.

b. Symbiosis Feasibility Analysis — Since our project involves a synthetic yeast–lactobacillus symbiotic system, we placed special emphasis on assessing whether the two species could coexist stably under co-culture conditions, avoiding system failure due to resource competition or metabolic incompatibility. Through a constraint-based co-culture modeling approach, we verified that the symbiotic interaction enhances the growth of the engineered yeast and supports the overall functional feasibility of our design.

By integrating predictive modeling and experimental validation across these two dimensions, we were able to comprehensively identify potential risks in our system and establish a robust scientific and safety foundation for subsequent experimental implementation.

(1) Metabolic Toxicity Analysis

a. We imported the genome-scale metabolic network of Saccharomyces cerevisiae from the BiGG Models database. We integrated six exogenous functional modules—adsorption, degradation, therapy, and their combinations—each encoding specific proteins. Using Python’s COBRApy package, we evaluated how the expression of these foreign genes affects the metabolic homeostasis of the chassis cell.

b. Flux Balance Analysis (FBA), a stoichiometry-based metabolic modeling method, enables quantitative assessment of how gene perturbations, heterologous pathways, or environmental changes influence cellular energy metabolism and growth. Through FBA, we calculated the ATP/GTP demand for protein synthesis and secretion across different expression levels and used growth rate variation as an indicator of metabolic toxicity. The results showed that within the normal expression range (10-5–10-4), the host’s growth rate decreased by less than 5%, indicating strong metabolic compatibility of the designed circuits.

c. To ensure the robustness and practical reliability of our results, we performed a sensitivity analysis on the uncertain biomass parameter—the energy cost associated with protein secretion. The results demonstrated that our model is highly robust and biologically meaningful.

d. Overall, this analysis defined the physiological safety boundary of our genetic design and provided a quantitative basis for the subsequent development of the project’s safety module.

(2) Symbiosis Feasibility Analysis

a. To mitigate the metabolic toxicity described above, our team actively explored the design of biosafety modules and proposed a yeast–lactobacillus symbiotic system as a novel Metabolic Safety Strategy. Through cross-species metabolic complementation, this system redistributes energy demands and metabolite fluxes, thereby alleviating the physiological burden on the engineered host at the systems level.

The imported model file is shown in the figure below.

b. As conducting in vivo experiments in humans would violate safety guidelines, we instead constructed a metabolic coupling model of yeast and Lactobacillus using Python and the COBRApy framework. We further introduced an FVA-constrained model to analyze metabolic flux distribution and maintenance energy demand (ATPM) under symbiotic conditions. The results showed that bidirectional exchange of key metabolites—such as lactate, amino acids, and riboflavin—significantly reduced the yeast’s maintenance energy burden and mitigated the metabolic toxicity caused by heterologous gene expression.
The mathematical formulation of our model is presented as follows.

c. Under multiple functional perturbations, the symbiotic system consistently exhibited higher growth rates and greater metabolic stability than monoculture models, demonstrating a safety-regulatory mechanism of “metabolic complementation – energy sharing – homeostasis enhancement.”

In summary, the symbiotic system not only proves physiologically feasible but also functions as a metabolic safety module that can be embedded into synthetic circuit design. This framework provides future iGEM teams with a quantitative and verifiable paradigm for building biologically safe and robust engineered systems.

1.2 HP exploration

Members of our human practice group interviewed Professor Wang Yongming from Fudan University. Through the interview, we learned about the risks of metabolic toxicity. After further in-depth exploration, we understood the solutions to reduce metabolic toxicity. The following are the guidance and research work we have obtained：
In the early days, genetic engineering experiments tended to be regarded as being successful once a certain level of expression of an introduced gene was achieved. However, this perspective, for the most part, ignored any physiological changes to the host organism that might have occurred as a consequence of the introduction of the foreign DNA, which is called “metabolic burden”. The "metabolic burden" refers to a phenomenon in which the induced expression of foreign genes in recombinant host cells or organisms typically consumes a large amount of host cell resources and brings adverse effects to the host^[4]. Due to metabolic burden, the biochemistry and physiology of host cells may undergo significant changes. For instance, this can impede the expression of endogenous essential genes within the cells, leading to a decrease in cell growth rate or a reduction in the synthesis of their own protein. On an industrial scale, these are reflected in low production titers and the loss of newly acquired traits, especially during fermentation runs^[5]. The main mechanisms by which overexpression of foreign genes causes metabolic burden are the stringent response due to amino acid/charged tRNA starvation^[6] and the increase in the number of misfolded proteins caused by translation errors leads to an increase in the stress on intracellular chaperone proteins and proteases, thereby activating heat shock and nutrient starvation responses^[7]. To address the adverse effects of metabolic burden, the following solutions are proposed:
a. Use low, rather than high, copy number plasmid vectors to avoid the host cells wasting resources in synthesizing unneeded antibiotic resistance marker gene products.
b. Use strong but regulatable promoters to control. The promoter of the target gene is in a closed state during the cell growth stage. And at the induction stage, the promoter is in an open state.

1.3 Experimental and circuit design measures

For us, the key point of metabolic toxicity lies in ensuring the growth of the target yeast while resolving the conflict between yeast growth and gene expression.
In terms of experiments and circuit design:
(1) Firstly, our Saccharomyces cerevisiae is inherently deficient in Ura and His. Using nutritional defect screening not only ensures safety but also reduces the competition for nutrients by non-target bacteria.
(2) Secondly, our symbiotic system mainly involves lactic acid bacteria providing nutrients to the yeast, which can also reduce metabolic toxicity.
(3) In our therapeutic circuit, the yeast has enhanced expression of SOD1 and CTT1. Although the signal peptide enables their secretion, the residual proteins remaining in the cells can reduce the oxidative stress caused by toxicity. We will later study the secretion efficiency of the signal peptide (the ratio of target proteins inside and outside the cell to explore the balance between therapeutic efficiency and attenuation effect)
(4) Finally, we selected a dynamic regulation system to control some exogenous genes. By using the gal promoter to control the expression of downstream target genes, while the chassis is Saccharomyces cerevisiae with gal80 knocked out. This can inhibit the expression of exogenous genes in the early growth stage of Saccharomyces cerevisiae (when it is in the growth phase), and concentrate on expressing the target genes in the later growth stage^[2].
(5) Furthermore, the relevant suggestions from the Advisor also pointed out issues regarding the orthogonality of the pathways. We drew inspiration from the regulation of the gal promoter. The CRISPR/dCas9 system only functions in a high-sugar environment during feeding, and at this time, the dynamic regulatory system in the fourth point inhibits the expression of the adsorption module. Thus, the functions of the degradation module and the adsorption module become orthogonal, and the expression of different modules initially has a temporal sequence.
(6) We constructed plasmids for some of the genes and integrated the remaining genes into the genome using CRISPR/Cas9. This hybrid system is conducive to achieving the optimal balance among stability, flexibility, and metabolic burden^[3].

2. Product safety

2.1 Product form

(1) Strain selection

In the strain selection process, Safety & Security has always been our top priority. All candidate chassis bacteria were rigorously compared: Escherichia coli K-12, although having mature tools, lacks sufficient safety; Nissle 1917 has limited public acceptance; some Bacteroides and Clostridium species have colonization advantages but may have controversies regarding toxicity or pathogenicity; Lactobacillus plantarum, as a food-grade probiotic, has good safety and tolerance, but has low expression efficiency and is not suitable for engineering; Saccharomyces cerevisiae, due to its GRAS certification, is widely used in food and medicine, ensuring safety, but has limited intestinal colonization. Our initial research led to the first conclusion: no single strain can fully meet the requirements. However, E. coli Nissle 1917 and Saccharomyces cerevisiae perform well in terms of safety and engineering tools, while Lactobacillus plantarum has unique anti-inflammatory and adsorption advantages.
After the engineering cycle, to balance Safety & Security, we finally chose the food-grade symbiotic system solution, consisting of Saccharomyces cerevisiae and Lactobacillus DT88. The yeast is responsible for executing the complex synthetic pathways and therapeutic modules, while DT88, as a food-grade probiotic, not only enhances the safety and stability of the system but also has natural microplastic adsorption and intestinal probiotic effects. The interdependence of the two reduces the possibility of the single strain's uncontrollable spread in the environment, which is more in line with biosafety requirements.
In the specific design, we additionally introduced multiple safety strategies, including an inducible expression system to prevent the loop from going out of control, an environment-dependent mechanism to ensure that the strain cannot survive long-term outside the host, and an exogenous controllable removal module to ensure that the engineered bacteria can be quickly removed when needed. And there is also spore prevention for the yeast chassis bacteria. These measures ensure that the engineered bacterial system complies with the "controllable, limited, and removable" biosafety principles, and the details will be explained in other parts.

(2) Product preservation method

Traditional live probiotic and engineered microbial products are highly sensitive to environmental conditions such as temperature, oxygen, light, and humidity. In traditional liquid or refrigerated storage, the number of live bacteria decays rapidly, and a costly cold chain system is required to maintain the efficacy and safety of the products. The interruption of the cold chain not only affects the quality of the products but may also accelerate the inactivation and deterioration of microorganisms.
Lyophilization is a mature industrial solution to address these challenges. This technology is carried out under low temperature and vacuum conditions, effectively removing moisture through ice sublimation, avoiding the thermal damage and oxygen poisoning caused by traditional hot drying. The product after freeze-drying is transformed into a lightweight and stable solid powder, significantly extending the shelf life. This powder form of the product can be directly transported and stored at room temperature without the need for a cold chain, greatly improving the accessibility and application scope of the product, especially for remote areas lacking stable low-temperature storage facilities, which constitutes an important application safety advantage.
Before freeze-drying, the strains are first microencapsulated by the alginate-chitosan (Alginate-Chitosan, A-C) system. Microencapsulation plays a triple role in preservation and delivery:
a. Physical protection: The A-C capsules act as the first physical barrier, protecting the symbiotic strains inside from ice crystal formation and dehydration stress during freeze-drying;
b. Enhanced mechanical stability: The chitosan-alginate complex forms through electrostatic interactions, showing higher mechanical stability, lower drug leakage rate, and less burst release compared to pure alginate capsules. This mechanical integrity is crucial for subsequent freeze-drying and storage;
c. Targeted delivery: The A-C coating gives the product the ability to resist degradation by gastric acid and bile salts, ensuring that the live bacteria can be released in a controlled manner in the target sites, such as the small intestine or colon, after passing through the stomach.
The preservation strategy shifts the safety paradigm of the product from "biological activity" to "biological dormancy". The greatest safety risk of live microbial products stems from their metabolic activity and reproductive ability. Through freeze-drying, the product's state shifts from "potential biological risk" to "physical and metabolic locked solid biological material". This forward-looking safety design shifts the focus of safety from post-treatment clearance or inhibition to pre-treatment activity locking, providing a more reliable guarantee for the long-term safety of the product.
In addition, the freeze-dried powder form provides physical isolation, while metabolic locking eliminates the risk of horizontal gene transfer of live bacteria to the environment or foreign bacterial communities during storage and transportation, maximizing the biological safety during the storage period.

2.2 The use of the product

When the product is used, ensuring that the yeast can function properly in the human body is also an important aspect of safety. Our team mainly considers this issue from two main directions: Firstly, the strain needs to survive normally in the human body. Secondly, the target function of the strain must be guaranteed in the human body.

(1) Strain life support

a. Symbiotic system support
One of the core advantages of this product lies in its unique dual-bacteria symbiotic system. This system is composed of genetically engineered Saccharomyces cerevisiae and probiotic Lactiplantibacillus plantarum DT88. This design is not a simple stacking of strains, but rather the creation of a miniature ecosystem. The primary purpose of this design is to provide key life support for the engineered yeast in the complex intestinal environment, thereby directly enhancing the Safety and Security of the product. We mainly investigated the advantages of this system in terms of "metabolic mutualism" and "stress tolerance", and also analyzed the potential risks and provided solutions.

a.1 Nutrient mutualism
In an environment rich in nitrogen sources in the intestinal tract, Saccharomyces cerevisiae actively secretes various amino acids, providing essential growth factors for amino acid-deficient Lactobacillus plantarum ^[8]. This targeted nutrient supply significantly promotes the growth and survival of Lactobacillus plantarum in a co-culture system, with its biomass being much higher than that in a single culture condition^[9]. In return, Lactobacillus plantarum can decompose complex carbohydrates (such as lactose) that yeast cannot utilize and convert them into monosaccharides like glucose, providing additional energy sources for yeast. This two-way nutrient supply creates a stable "nutrient support complex" in the resource-variable intestinal environment.
① Safety and Effectiveness Goals: Enhance effectiveness: This mutually beneficial symbiotic relationship ensures that our engineered bacterial system can maintain stable biomass and metabolic activity even in the fierce competition of the native intestinal microbiota. This directly guarantees the continuous and stable secretion of therapeutic proteins and ensures the reliability of product functionality.
② Endogenous biological containment: More importantly, this "nutrient interlock" mechanism builds a survival-dependent relationship. It firmly binds the two strains together, significantly reducing the possibility of a single engineered strain (especially yeast) surviving independently after accidental leakage into the external environment. Once deprived of the nutritional support of Lactobacillus plantarum, the survival ability of the engineered yeast will be significantly limited. This is an important endogenous containment link in our biosafety design, aiming to minimize environmental release risks from the source.
a.2 Stress tolerance
① The advantage in the intestinal environment - bile salt protection^[10]: Saccharomyces cerevisiae can secrete extracellular peptide substances known as "reactivation factors". In the upper part of the intestine, high concentrations of bile salts have a strong killing effect on most oral microorganisms. These "reactivation factors" act like a "protective shield", significantly protecting the coexisting lactic acid bacteria from the stress damage caused by bile salts, ensuring that the entire symbiotic system can safely pass through the stomach and the upper part of the small intestine in an alive form.
Safety and effectiveness goals: The purpose of this mechanism is to ensure that the engineered bacteria with an "effective dose" can reach the target action area in the lower part of the intestine. This directly overcomes one of the biggest challenges of oral live bacterial preparations - low bioavailability, thereby ensuring the therapeutic effect of the product. Of course, we continue to optimize tolerance in that direction.
② The advantage in the intestinal environment - ethanol detoxification^[11]: In the anaerobic environment deep in the intestine, yeast undergoes alcohol fermentation, producing ethanol that also inhibits its own growth. Research has found that co-culturing with Lactobacillus plantarum can significantly improve the ethanol tolerance of Saccharomyces cerevisiae.
Safety and effectiveness goals: The purpose of this mechanism is to extend the functional lifespan of the engineered bacteria in the intestine. By alleviating the self-toxicity of ethanol, the symbiotic system can maintain higher activity and population density in the intestine for a longer time. This not only means a more lasting therapeutic effect but also enhances the stability of the system, avoiding the interruption of the therapeutic effect due to the premature collapse of the population, ensuring the predictability and safety of the entire treatment cycle. It also more clearly demonstrates the dependence of the two, reducing the potential risk of the engineered yeast cells leaving the symbiotic system and causing harm to the human body.
③ The advantage in the intestinal environment - inhibiting immunity^[9]: Anti-inflammation itself is a therapeutic effect, but this effect also reduces the risk of the symbiotic system being attacked by the immune system. Studies have confirmed that the yeast-lactobacillus co-culture system can synergistically significantly increase the production of IL-10 and inhibit TNF-α. This indicates that this system can actively push the immune response towards a tolerant state.
Safety and effectiveness goals: This mechanism is an extension of the thinking of anti-inflammatory assistance, which involves the analysis of the immune microenvironment around the symbiotic system. Enhancing its anti-inflammatory effect is also to reduce the stress of the immune response on the symbiotic system, and the continued survival of the strain can also continue to exert the anti-inflammatory effect, forming a positive feedback loop. Of course, this is based on the symbiotic system, and if the engineered yeast cells leave the symbiotic system in the intestine, they will be significantly stressed.

a.3 Security considerations and risk control in symbiotic systems
Although symbiotic systems offer significant functional advantages, their complexity also introduces risks that must be managed with caution. Our design has always taken the following risk points as the core consideration and has adopted multi-level mitigation strategies. See Attachment: Security Considerations and Risk Control forSymbiotic Systems.
b. Anti-gastric acid bile salts
Following the iGEM "Safe-by-Design" concept, we recognize that a responsible engineering project requires a safety system that is actively constructed and multi-layered with redundancy. The design of the alginate-chitosan microcapsules is the first and most crucial physical defense line in our entire safety framework.

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b.1 Ensure targeted delivery and biological containment
Although the symbiotic system we designed enhanced the survival ability through its internal "stress tolerance" mechanism, to safely apply a living, genetically engineered organism to the human body, the problem of protection during oral delivery must be solved. The primary challenge faced by oral live bacterial preparations is the stressful environment in the upper part of the digestive tract. The strong acidity in the stomach (pH 1.5–3.5) and the bile salts in the upper part of the small intestine not only cause the live bacteria to lose their viability, but more importantly, we need a physical barrier to ensure that the engineered bacteria do not interact with non-target environments before reaching the target action area, thereby minimizing the risks of accidental exposure and environmental release to the greatest extent.
To address this challenge, we designed and verified a dual-layer composite microcapsule system. This system consists of a sodium alginate core and a chitosan shell, using the electrostatic interaction between the two GRAS-level materials to form a denser and more stable physical barrier that can resist stomach acid and bile salts.
*PS:
Anti-acid mechanism: In the highly acidic environment of the stomach (pH 1.5–3.5), the negatively charged sodium alginate undergoes protonation to form insoluble alginate, and its gel network contracts and becomes denser, thereby physically encapsulating the internal bacteria firmly. At the same time, the outer chitosan, due to the protonation of its amino groups, acquires positive charges and forms a stable polyelectrolyte complex with the alginate, further enhancing the structural integrity and acid resistance of the capsule, effectively resisting the erosion of gastric acid^[12].
Anti-bile salt mechanism: After entering the small intestine, the high concentration of bile salts acts as a biological surfactant and can damage the microbial cell membranes. The dense composite microcapsules provide an effective physical barrier to prevent the penetration of bile salts, protecting the internal bacteria and ensuring their survival and reaching the lower part of the intestinal tract^[13]. Through an in vitro experiment simulating the gastric environment, we rigorously verified the protective ability and biological containment effectiveness of the microcapsules. The experimental results clearly demonstrated that this composite microcapsule could effectively resist the erosion of simulated gastric fluid and successfully contain the live bacteria within the capsule. Its pH-responsive design ensured that the microcapsule would disintegrate only when it reached the specific pH environment of the intestine, meaning that the GMM could only be released at the predetermined application site. This "targeted release" mechanism significantly reduces the possibility of its colonization in non-target areas or being maliciously extracted and misused, reflecting our careful consideration of the Security aspect of the project.

b.2 A safety commitment based on absolute harmlessness
Our design starts from the first principle of material selection: absolute safety. To minimize risks from the very beginning, we systematically investigated various biocompatible materials and strictly limited ourselves to the GRAS (Generally Recognized as Safe) level.
We learned that both are natural polysaccharides, having decades of safe application history in the food and medical fields. They are non-toxic and non-irritating to the human body, and their biodegradable products are also safe. This choice ensures that our physical containment system itself does not introduce any new chemical or biological safety risks, fully meeting the basic requirements for project safety set by iGEM.
*PS:
Sodium Alginate: This is a polysaccharide extracted from natural brown algae and is recognized as a GRAS substance by the US Food and Drug Administration (FDA). It is widely used in the food industry as a thickening agent and stabilizer, and is also commonly used in the medical field for wound dressings and drug delivery. It has good biocompatibility, cannot be digested or absorbed by the human body, and will eventually be excreted through feces, with no toxic side effects^[14].
Chitosan: It is a natural polysaccharide obtained by deacetylation of chitin from the shells of crustaceans such as shrimp and crab. Chitosan also has excellent biocompatibility, biodegradability, and extremely low toxicity. It has been widely studied and applied in food, cosmetics, and drug delivery systems^[15].
At this point, we have completed the fabrication of the "physical armor" for the strain. Our ultimate goal is to combine this external physical containment strategy with our internal genetically engineered containment strategy to form a dual-protective system in order to address more complex security challenges and maximize the safety of users, communities, and the environment.

(2) Strain safety effect

Engineered bacteria can survive stably and function in the intestinal tract, and also ensure safety and harmlessness during actual action, bringing positive effects to the host.
a. Specificity and harmlessness
The degradation enzymes secreted by the enzyme degradation module, such as PETase and MHETase, have clear substrate specificity and only act on the microplastic structure, without degrading the host's proteins or polysaccharide components.
The products after microplastic degradation are small molecule monomers or oligomers, without toxicity, and have no adverse effects on the host or the intestinal microbiota.
The adsorption module uses the HFBI surface to display protein to bind to microplastic particles, and the target object is limited to hydrophobic surfaces, without conflicting with the attachment sites of intestinal tissues or normal microbiota.
b. Reduce potential damage caused by microplastics
The synergistic effect of the enzyme degradation and adsorption modules can accelerate the clearance of microplastics, shorten their residence time in the intestine, avoid long-term physical stimulation and chronic inflammation, and at the same time reduce the risk of oxidative stress and barrier disruption caused by them, and reduce the burden on the intestinal environment.
c. Promote intestinal health and protection
The SOD1, TFF3, and CTT1 secreted by the treatment module, respectively, undertake the functions of eliminating free radicals, repairing the mucosal barrier, and decomposing peroxides. These molecules can work in synergy with the host's natural defense factors to alleviate oxidative stress and inflammation, not only being harmless but also protecting intestinal homeostasis and reducing further harm to the bloodstream.
d. Stable colonization and immune friendliness
Lactobacillus has acid resistance, bile salt resistance, and colonization ability. While ensuring the safe delivery of yeast to the intestine, it can form protective biofilms locally, and it can symbiotically coexist with yeast and metabolize complementarily, enabling the engineered microbiota to exist stably and express normally in the intestine. The formed system can jointly adsorb microplastics, reduce their contact with the intestinal mucosa, and exert anti-inflammatory and immune regulatory effects through the secretion of lactic acid, amino acids, etc., which is beneficial and harmless to the human body.

(3) Disposal of products

a. Safety Route Review
To further ensure safety, we began to explore biological containment strategies. We believe that the safest and most effective method is through a mode: signal-controlled sterilization. It involves sensors and effectors, just like the reflexes in the human body, simple yet highly effective. Our team conducted a partial systematic review of sensors and effectors based on the requirements of our project. Although they come from different platforms, they can still provide ideas for our biological containment strategies.

a.1 Sensor
Based on our project application scenarios, we classify the input signals into two different types: 1) Input signals that are regulated by the inherent conditions of the body - "passive" input signals, 2) Input signals that require external conditions to be applied for regulation - "active" input signals.
a.1.1 "Passive" input signal
① The temperature control system of the engineered bacteria utilizes the inherent temperature difference between the internal and external body environment. This enables the triggering of cell death at lower external temperatures. Advantages: It utilizes the natural temperature difference and does not require manual intervention. Disadvantages: It needs to consider the possibility of bacterial escape during the transportation process in the presence of low temperatures and hot weather. For our project, this issue can be addressed. Our product is preserved through freeze-drying technology, ensuring that it will not be abnormally activated before entering the body. Regarding the hot weather issue, we believe that the environment where the excretions are located will not exceed the body temperature. Of course, this reminds us to pay attention to the temperature threshold for activation. In addition, the method of activation is also diverse. For example, the NMU-China 2024 and Athens 2022 teams have all applied this sensing method using different technologies in the safety module^[16][17].

② Besides temperature, we also referred to other lines that can sense the differences in internal signals. We applied the following ideas to our degradation and adsorption modules, which are in line with our previous engineering cycle goals.
— The deep part of the human intestinal tract is a typical hypoxic/microoxic environment, while the external environment (air, water) is oxygen-rich. This huge oxygen concentration gradient provides an excellent opportunity for designing safety switches. The PTSH-Taiwan 2023 team utilized an oxygen-sensitive promoter in their project to control the expression of toxins, ensuring that the engineered bacteria could be effectively eliminated when they were in the oxygen-rich environment outside the intestine.
— SZU-China 2021 developed a glucose starvation system involving the use of glucose starvation receptors. When glucose is absent, these receptors will induce the expression of downstream genes. This system uses the inherent difference in glucose concentration between the intestinal tract and the external environment to induce bacterial death in the external environment.

③ The phosphate receptor is a receptor proposed by SZU-China 2021. It utilizes the difference in phosphate concentration between the inside and outside of blood vessels to prevent engineered bacteria from entering the circulation through minor vascular ruptures in the intestinal tract during inflammation. We believe that this system ingeniously solves the problem of preventing bacteria from entering the bloodstream, which is a problem that other teams have rarely considered. Regarding the issue of blood entry, we currently have a preliminary blocking effect through symbiosis to enhance the intestinal mucosal barrier^[18][19].

a.1.2 "Active" input signal
These two types of signals can fully exert the stabilizing effect of the artificial control circuit and can effectively control the intensity of the input signal for testing, ultimately achieving the optimal result. In terms of nature, they include physical signals: light, electricity, temperature, etc., and signal perception.
Here, taking the light receptor as an example, SZPT-CHINA 2021 uses a simple promoter to sense blue light. This regulatory system is simple and stable. In shady areas, when the light intensity is insufficient, it may be necessary to artificially apply blue light to induce death. Chemical signals are mostly regarded as "drug" signals. Our NMU-China team has discussed the use of "drugs" in the safety module in previous editions of IGEN. This inspired us to compare the advantages and disadvantages of different "drug" signals in intestinal applications to obtain the best option.

a.2 Effector
a.2.1 Toxin-antitoxin,(TA) system

Toxin-antitoxin (TA) system is one of the most fundamental and classic tools for constructing "self-destruct switches" in living organisms. Such systems typically consist of a stable toxin that can disrupt key physiological processes of the cell, and an unstable antitoxin that can neutralize this toxin. By placing the expression of one or both components under the control of specific environmental promoters, the team can program the cells to survive only in the pre-defined ecological niche. The following reviews how previous iGEM teams have creatively applied various TA systems.
— CcdB/CcdA system: This system utilizes the CcdB toxin, which can lethally inhibit the DNA gyrase (an enzyme crucial for DNA replication). Its effect is neutralized by the CcdA antitoxin protein. For instance, the GEMS_Taiwan 2022 team employed this system^[20].

— Hok/Sok System: This is an I-type TA system. In this system, the toxin (Hok) is a protein that damages the cell membrane, while the antitoxin (Sok) is an unstable antisense RNA that binds to the mRNA of Hok, preventing its translation and promoting its degradation. For example, the Wageningen_UR 2021 team has used this system^[21].
— MazF/MazE system: This is a thoroughly studied type II TA system. Among them, MazF is a stable mRNA endonuclease that can cut cellular mRNA, while MazE is an unstable protein antitoxin that will be rapidly degraded by cellular proteases. For example, the Fudan 2020 team used this system^[22].

— VapD/VapX system: This is a relatively novel type II TA system. In this system, VapD is a ribonuclease toxin, and VapX is its protein antitoxin. The Vilnius-Lithuania 2021 team was the first to characterize and apply it in iGEM^[23].

— Holin/Antiholin System: Holin is a toxin protein that can "make holes" in the cell membrane. These holes allow other molecules (such as endolysins) to enter and degrade the cell wall. Antiholin is an antitoxin that can bind to Holin and inhibit its hole-making activity. For example, the Cornell 2020 team used this system^[24].

a.2.2 Hijacking Apoptosis
This strategy no longer relies on exogenous toxins, but instead precisely controls cell fate by activating the endogenous, highly conserved programmed cell death (apoptosis) pathway within eukaryotic cells.
— Bax is a core pro-apoptotic protein in the apoptotic pathway of mammalian cells. When expressed in yeast, Bax can target the outer mitochondrial membrane, form channels, disrupt mitochondrial function, and ultimately induce apoptotic-like death in the cells. The GreatBay-SCIE 2023 team envisioned in their project a self-destruct switch using Bax, for clearing the engineered yeast after completing its task. Their design concept is to induce the expression of Bax after the engineered bacteria leave the target environment, thereby triggering the endogenous death program^[25].

a.2.3 Targeted gene system
These effectors carry out "execution" by permanently modifying the genetic material of cells. Once triggered, there is almost no possibility of escape, representing the cutting edge of current biosecurity design^[26].
— CRISPR-based genome degradation: The CRISPR/Cas9 system is like a programmable "genetic scissors". By expressing Cas9 enzyme and one or more guide RNAs (gRNAs), multiple essential genes in the genome can be precisely cut. The large number of DNA double-strand breaks (DSBs) produced in a short period of time will far exceed the cell's repair capacity, leading to genome collapse and irreversible cell death.
AQA_Unesp in 2017 designed a more sophisticated light-controlled switch. They split the Cas9 protein into two inactive fragments and fused each with a photoreceptor protein (such as CRY2 and CIB1). Only under blue light irradiation can the two fragments reassemble into an active Cas9 enzyme, thereby cutting the targeted multiple bacterial essential genes (such as dnaN, rpoC, etc.), achieving precise and temporal control of the self-destruct program.

— Site-specific recombinase: The Cre/LoxP system is a representative example. The Cre recombinase can recognize two LoxP sites and irreversibly excise or reverse the DNA fragment between them. By placing the essential gene or its promoter between the two LoxP sites, the function of the gene can be permanently disrupted by inducing the expression of Cre. The PTSH-Taiwan 2023 team designed an extremely sophisticated double-layer logical switch for closely related yeast. This design decouples the two steps of "arming" and "firing" of the system in terms of time and space. In the intestine (a safe environment with high hydrogen sulfide), both Cre recombinase and the antitoxin are expressed simultaneously. The Cre enzyme permanently removes the terminator upstream of the toxin gene, completing the "arming", but the cells survive due to the presence of the antitoxin. When the cells leave the intestine (low hydrogen sulfide, high oxygen), the antitoxin stops expressing, and at the same time, an oxygen-sensitive promoter activates the expression of the toxin, completing the "firing", killing the cells. This strategy of using irreversible genetic modification for "state memory" greatly enhances the robustness of the system^[27].

a.2.4 Engineered Dependency
Unlike the active introduction of toxins, this strategy achieves passive containment by modifying the metabolic network of cells to make them dependent on a specific environment or symbiotic partner for survival.
— Conditional essential genes: The NEU-CHINA 2022 team did not use CRISPR as a direct lethal weapon, but rather regarded it as a tool for constructing a safe pathway. They utilized the CRISPR technology to replace the natural promoter of a yeast essential gene - the heat shock transcription factor HSF1 - with a copper ion-inducible promoter CUP1. Due to the much higher concentration of copper ions in the intestinal environment compared to the outside, the survival of this engineered yeast is tightly bound to the intestinal environment. Once it leaves, it will die because it cannot express the essential gene^[28].

— Reciprocal nutritional defect: The Calgary 2020 team successfully constructed a "metabolic lock" system consisting of two yeast strains. Strain A was modified to be a leucine nutritional defect strain, but it can over-secrete tryptophan; Strain B, on the contrary, is a tryptophan nutritional defect strain, but it can over-secrete leucine. These two strains can only "feed" each other and grow together when co-cultured. Once they escape into the environment and are diluted and separated, the individual strain will "starve" due to the inability to obtain the necessary amino acids. The evolutionary stability of this strategy is higher because cells need to escape through more difficult recovery mutations rather than simple gene inactivation mutations^[29].

b. The design of the stop switch in our project
After reviewing the above sensors and effectors, we gradually began to build the prototype of the safety circuit.
b.1 First-generation design: A temperature-sensitive switch for in vitro biological inhibition
The core objective of the first-generation design was to achieve in vitro biological containment, aiming to prevent engineered bacteria from entering the environment and causing potential ecological risks due to unexpected survival or horizontal gene transfer (HGT). To this end, we constructed a safety switch that could quickly initiate programmed cell death when the engineered bacteria completed their tasks within the host and left the host. According to our reviewed safety pathways, we believe that temperature is a relatively ideal sensing signal.
This design utilizes the stable physical temperature difference between the host's intestinal tract (approximately 37°C) and the external environment (lower than 30°C) as a signal. Driven by this temperature gradient, an "inside-off, outside-on" logic gate is achieved, which we call the Cold-Triggered Kill Switch. Its core is a reverse logic circuit: the heat shock promoter PHSP26 drives the expression of the repressor protein TetR to turn off the killing genes at 37°C; when the temperature drops to the external level, the expression of TetR is downregulated, thereby releasing the inhibition on the downstream killing module.
Inspired by the feedback from the effectors, we concurrently developed two killing effector modules, both targeting cellular genetic material:
Scheme 1 (Metabolic Blockade):
Through the Cre/LoxP system, the key gene ADE2 of the purine synthesis pathway is knocked out at low temperatures, thereby blocking DNA replication.

Scheme 2 (Genome Degradation):
Express the NucA nucleases of Pseudomonas aeruginosa directly at low temperatures to efficiently degrade genomic DNA.

Experimental and bioinformatics analyses have verified the feasibility of this design. However, although this design can effectively control leakage in vitro, it has a fundamental flaw: it lacks an in-body emergency clearance mechanism that is actively controlled by the user. Based on user practice and expert consultation feedback, this aspect is crucial for building patients' trust and sense of security.
b.2 Second-generation design: Drug-induced toxin-antitoxin switch
To address the lack of in-body control in the first-generation design, after review, we concluded that the drug signal has advantages for in-body control. In terms of effectors, the second-generation design introduces a drug-induced toxin-antitoxin (Toxin-Antitoxin) system, giving users the ability to actively remove engineered bacteria from the body when necessary. This system is based on the K1 killing toxin system of Saccharomyces cerevisiae. Among them, the unprocessed K1 toxin precursor protein contains an immune region inside, which can protect the host from harm and play the role of "antitoxin". The mature αβ heterodimer that is processed and secreted outside the cell is the "toxin", which can lyse the yeast cell wall and cause death.

The system's switch is controlled by exogenous arabinose: in the normal state, the system remains in a safe condition. When the user orally ingests arabinose, the inducible promoter pAra is activated, driving the efficient expression of the toxin module. Eventually, a large amount of mature toxin proteins is produced and secreted, whose killing effect exceeds the immune protection ability of the organism itself, thereby achieving precise and targeted elimination of the engineered bacteria in the body.
Literature research and preliminary experimental verification have confirmed the feasibility of this scheme. However, this system still has potential areas for optimization: Firstly, the arabinose promoter has the risk of background leakage expression; Secondly, the individual differences in the host's absorption and metabolism rate of arabinose may affect the stability of the induction efficiency. These issues will be the focus of our subsequent work.
b.3 Further prospects and outlooks
Based on the practical experiences of the two generations of designs, we identified the shortcomings of the safety circuits through comparison. Therefore, we have planned the future research direction, aiming to build a more rigorous, intelligent, and secure biological containment system.
b.3.1 The rigor and robustness of the safety switch: Regarding the leakage issue of the arabinose system, future work will focus on constructing more precise regulatory circuits, such as introducing toxin-antitoxin neutralization modules or designing logical "AND gates" that require dual signal inputs, to fundamentally prevent unintended toxin expression.
b.3.2 The uniformity and predictability of clinical applications: To overcome the impact of individual metabolic differences, subsequent research needs to explore standardized dosing schemes or screen and validate alternative inducers with clearer metabolic pathways and smaller individual differences.
b.3.3 The intelligence and autonomous regulation of the system: To enhance the precision of intervention, future work can integrate the quorum-sensing module into the safety system. This will enable the engineered bacterial population to autonomously activate the growth inhibition program when reaching a specific density threshold, achieving dynamic balance and avoiding excessive proliferation.
b.3.4 Multiple redundant biological containment strategies: To address extreme situations (such as the engineered bacteria breaking through the intestinal barrier), the final safety design should include multiple, orthogonal containment mechanisms. For example, on the basis of the existing killing switch, additional nutritional defect designs can be integrated to ensure that the engineered bacteria cannot get out of control in any environment.

c. Spore prevention

Although Saccharomyces cerevisiae belongs to BSL-1 microorganisms, its ability to form spores may lead to Release Beyond Containment, which is explicitly listed as a risk point in the iGEM safety policy^[30]. Spores have extremely strong environmental resistance, and once formed, they are difficult to effectively control^[31].
In our project, the engineered yeast will be excreted from the host after completing its in vivo therapeutic task. If these yeasts encounter specific nutritional stress (such as nitrogen source deficiency) in the external environment, they may initiate the spore formation process^[32]. This will bring two unacceptable high-level risks:
① Environmental release and ecological risk: Spores have high resistance to drying, ultraviolet rays, and common disinfectants and can survive for a long time in the natural environment. If the spores carrying the engineered circuit spread in the environment, it may lead to gene drift, causing unpredictable and long-lasting impacts on the local microbial ecosystem.
② Genetic information diffusion risk: Spores are dormant carriers of genetic material. Their diffusion is equivalent to the "out-of-control spread" of our engineered gene circuit in the environment, which increases the risk of horizontal gene transfer (HGT) and may lead to the unintended use or abuse of our synthetic biology tools.
To eliminate this risk, we used genetic engineering techniques to delete the core regulatory gene IME1 that controls spore formation, thereby depriving the yeast of the ability to form spores. IME1 is the "master switch" for spore formation, functioning as a central processor that can integrate various signals (primarily nutritional status) from both inside and outside the cell and make the final decision on whether to initiate spore formation. Its absence will prevent diploid yeast from forming spores under any induction conditions, without affecting mitosis and normal metabolism. We chose to delete IME1 for the following reasons:
① Absolute core position: As the highest regulator of the spore formation signaling pathway, deleting IME1 can block the entire process from the very top, ensuring that the spore formation program cannot be initiated regardless of changes in the external environment^[33].
② High specificity, no off-target effects: The function of IME1 is highly specific, and its main role is to initiate meiosis. Studies have confirmed that deleting this gene will not have a significant negative impact on the yeast's normal mitosis (i.e., normal growth and reproduction) and other core metabolic functions^[34]. This ensures that our engineered bacteria, when exerting therapeutic effects, will not have their biological properties and stability affected.
③ Permanent and stable strategy: The gene knockout achieved through genome editing is permanent and genetically stable. This means that once the construction is successful, all descendant strains will inherit the "no spore" characteristic, without the need to worry about functional restoration or escape, and there will be no new safety issues in the future.
We have designed a set of schemes using CRISPR/Cas9-mediated homologous recombination to replace the coding region of IME1 with a fragment carrying His and Ura, and ensure the successful knockout through resistance screening and PCR verification.
Literature studies have proved that this strategy can eliminate the ability to form spores while maintaining the biological stability and application value of the strain. In subsequent experiments, we will place the modified yeast and the wild-type yeast in standard spore formation media (such as potassium acetate medium) and observe them under a microscope for several days. The wild-type should be able to see typical tetrad spores, while the modified strain will completely fail to form any spore structure.
In conclusion, this measure effectively avoids the risk of spore spread, ensuring that our chassis strain meets the strict biological safety requirements of iGEM.

3. Part and Material Safety

3.1 Overview

(1) Our Basic Part

We designed 20 basic parts in total this year, integral to our whole cycle.

(2) Our Composite Part

This year, we designed a total of 13 composite components, each assembled from different Basic Parts, forming the core of the circuit system.

3.2 Parts

(1) Toxic protein

a. K1 Killer Toxin and NucA
The mature K1 toxin is an ionophoric protein toxin that kills sensitive cells by forming pores in the plasma membrane. The K1 killer preprotoxin is a precursor protein that also functions as an antitoxin, protecting the host cell from the mature K1 toxin.
The nucA gene from Serratia marcescens encodes a non-specific endonuclease that degrades both DNA and RNA, often used as a suicide gene in biocontainment circuits.
These components form the core effectors of our “self-destruct switch,” designed to enhance the project's biological containment capabilities. Their mechanism of action exhibits no known toxicity to mammalian cells. Furthermore, their expression within our circuit is tightly regulated by conditionally activated promoters (such as the temperature-sensitive promoter pHSP26 or the arabinose-inducible pAra). This ensures system safety under non-induced conditions and, due to their non-novel, low-risk nature as toxins, poses minimal risk of malicious misuse.

(2) Functional protein

a. Fast-PETase and MHETase:
These two enzymes originate from non-pathogenic microorganisms and serve as core functional proteins for degrading microplastics. They exhibit high substrate specificity, acting exclusively on the chemical bonds of plastic polymers such as PET or MHET, without degrading biological macromolecules like proteins or polysaccharides within the gut. Consequently, they perform their degradation function while remaining completely safe for both users and the gut microbiota. As environmental remediation tools, they lack the potential for weaponization or malicious disruption, presenting low dual-use risks.
b. SOD1,CTT1 and TFF3:
These proteins are key therapeutic components in our project. SOD1, CTT1 (catalase), and TFF3 (trifolium factor) function as antioxidants and intestinal mucosal repair agents, respectively, benefiting the host. Derived from safe species or homologous to endogenous human proteins, they exhibit excellent biocompatibility with no known toxicity or immunogenicity risks. These therapeutically functional proteins carry no dual-use risks and cannot be repurposed for malicious purposes.

(3) Genetic Engineering Tools

a. dCas9:
dCas9 is an RNA-guided DNA-binding protein that lacks nuclease activity and serves as a modular platform for targeting specific DNA sequences.Although dCas9 loses its ability to cleave DNA, but fully retains its function of binding to specific DNA sequences under the guidance of single-guide RNA (sgRNA). This characteristic makes it a modular DNA-binding platform.In this project, the platform targets BAR1 to enable MXI1 to inhibit gene transcription. Unlike traditional CRISPR/Cas9, dCas9 is solely used for gene expression regulation (CRISPRi) without cleaving DNA, thus eliminating the risk of gene drive.
b. Cre Recombinase
Cre is a site-specific recombinase targeting lox sequences, capable of recognizing multiple variants (loxP, lox66, lox71, lox2272, etc.) and cleaving or inverting the intervening DNA sequences depending on their orientation. Literature and reviews indicate that Cre exhibits comparable recognition efficiency for single-mutant sites (e.g., lox66 or lox71) to that of wild-type loxP. However, lox72—generated by recombination between lox66 and lox71—exhibits significantly reduced affinity for Cre, thereby preventing further recombination^[35].
Additionally, the Cre recombinase sequence we used is BBa_K1680007, a yeast codon-optimized recombinase.
Based on the above, the function of Cre recombinase is strictly confined to performing a single, irreversible excision of a gene fragment (such as knockout of ADE2). It serves as a controlled “genetic scalpel” rather than a self-propagating element.
To sum up, the powerful potential of gene editing technology, along with an explanation of the project's mitigation measures, such as not publicly releasing key gRNA sequences, conducting bioinformatics screening on all designed sequences, and implementing strict physical and digital asset access controls within the laboratory.

3.3 Materials

(1) Physical containment Materials

a. Sodium Alginate and Chitosan
Sodium alginate and chitosan form the core of our physical containment strategy. Both are U.S. FDA-certified “Generally Recognized as Safe” (GRAS) substances, widely used in the food and pharmaceutical industries, non-toxic, and biodegradable. In this project, they are utilized to construct microcapsules, providing an effective physical barrier for engineered bacteria. Far from posing a risk, this constitutes a proactive measure to enhance project safety. It effectively prevents the release of bacterial strains into non-target areas, thereby elevating the overall safety and security standards of the project. For specific information, please refer to “Product Safety - Anti-Gastric Acid and Bile Salt Module.”
b. Chemical Inducers
Arabinose is a naturally occurring sugar and a safe food ingredient. It is scarcely absorbed by the human body and exhibits extremely low utilization within the body. In our project, it is suitable for use as an “active” input signal to trigger a user-controlled “self-destruct switch.” Its safety lies in providing users with a reliable, harmless means to actively clear engineered bacteria from their bodies. This “drug”-induced mechanism grants users or physicians ultimate control over the engineered bacteria's lifecycle. This serves as a positive security feature, ensuring the biosafety and controllability of the induction process within engineered Saccharomyces cerevisiae^[36].

(2) Target Material

a. PET
Microplastic powder is the target material for processing in our project, not a project byproduct. We have identified key risks associated with handling microplastics (particularly dry powder) during experiments, such as inhalation hazards and electrostatic dispersion. To ensure personnel safety, all relevant operations are conducted within chemical fume hoods or biosafety cabinets. Mandatory use of personal protective equipment (PPE), including N95 respirators, safety goggles, and gloves, is enforced to eliminate exposure risks. For details, please refer to the Microplastics Laboratory Safety section.

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