Prioritizing Lab and Project Safety

Our team places the highest priority on safety in both our laboratory work and our project design. We strictly follow the biosafety regulations of the Taiwan Centers for Disease Control (CDC) and the guidelines of Chung Shan Medical University. Before starting wet lab work, every member of our team received detailed biosafety and laboratory safety training. We always use Personal Protective Equipment (PPE) such as lab coats, gloves, and protective eyewear. We also follow good microbial practice, careful sterilization, and proper waste management procedures.

To further strengthen safety, we worked closely with our PI and our institutional biosafety officer. Together, we reviewed our protocols and made improvements where needed. This ensures that our project develops in a responsible way and that our laboratory remains a safe environment for everyone involved.

Ensuring Safety

Identifying Possible Risks

Our project engineers Lactobacillus spp. (a probiotic bacterium) to produce and secrete human anti–IL-23 single-chain variable fragments (scFv). IL-23 is linked to autoimmune diseases such as ulcerative colitis and psoriasis.

At the current stage, the risks are minimal because:

• Lactobacillus spp. are classified as BSL-1 organisms, safe for lab work.
• All experiments are limited to in vitro conditions.

However, we identified some possible risks for future applications:

• Unexpected immune reactions to the scFv when used in humans.
• Horizontal gene transfer if plasmids were to move to other bacteria.
• Public misunderstanding of using engineered probiotics as therapeutics.

How We Reduce These Risks

To minimize these risks, we:

• Use a plasmid-based expression system, reducing genome-level changes.
• Select only well-characterized probiotic strains (L. acidophilus, L. rhamnosus).
• Avoid virulence factors and antibiotic resistance markers that could increase hazards.
• Work strictly under BSL-1 containment, with supervision and documentation.
• Communicate openly that our project is at the research stage, not intended for human use at this point.

Our Experiments

Our design involves cloning the optimized anti–IL-23 scFv sequence (based on ustekinumab variable regions) into the plasmid pLEM415-IdhL-mRFP1, which we introduce into Lactobacillus acidophilus by electroporation. We confirm secretion and expression through Western blot and ELISA.

To optimize production, we compare culture conditions using different carbon sources (glucose, glycerol, lactose). For functional tests, we use a Caco-2 transwell model that mimics the human intestinal barrier. We stimulate inflammation with IL-23 and test whether our engineered bacteria or their supernatant can help restore barrier integrity. We measure this using:

• TEER (transepithelial electrical resistance) for barrier function
• qRT-PCR for gene expression of tight junction proteins
• Immunofluorescence staining for Claudin-8 (CLDN8)

All of these experiments are done in vitro under safe and controlled lab conditions.

Expert Guidance

We consulted our supervisor and institutional biosafety officer, Dr. Yi-Hsien Hsieh (hyhsien@csmu.edu.tw), who confirmed that our project is safe at the BSL-1 level. We also engaged with external experts and stakeholders to review our safety strategy. Their feedback helped us refine our design and communication approach, ensuring that risks are minimized and responsibilities are clear.

Our Lab and Training

Our wet lab work is carried out in certified BSL-1 laboratories at Chung Shan Medical University. All members were required to complete safety training sessions led by supervisors and laboratory technicians. The training covered:

• Lab entry rules and PPE usage
• Sterile technique and good microbial practices
• Disinfection and sterilization
• Emergency procedures
• Waste management
• Safe use of chemicals and reagents

To maintain safety:

• At least two team members must be present in the lab during work.
• Eating, drinking, and storing personal items are prohibited.
• Closed shoes, long sleeves, and tied hair are required at all times.
• Lab coats, gloves, and safety glasses are mandatory.

By following these rules, we keep both the team and the lab environment secure.

Waste Management

Waste handling is a critical part of biosafety. We separate biological waste from uncontaminated waste, and all biological waste is autoclaved before disposal. Liquid and solid wastes are treated separately to prevent contamination.

Chemical waste is handled according to institutional rules and stored securely until disposal by trained personnel. By following these measures, we make sure that nothing hazardous leaves the laboratory.

Risk Management Actions

We took additional steps to strengthen our safety system:

• Project-specific biosafety training for handling Lactobacillus and genetically modified organisms.
• Consultation with biosafety experts to confirm that our work follows regulations.
• Countermeasure planning in case of accidental exposure, based on the safe and non-pathogenic nature of Lactobacillus.
• Responsible communication plan to explain clearly that our project is not intended for direct use in humans at this stage.
• Safer reagents: For example, using SYBR Safe instead of ethidium bromide to reduce chemical hazards.

Future Applications

In the future, if our project develops into a therapeutic product, additional safety measures will be required. For example:

• Preclinical testing in animal models would be needed before human use.
• Clinical trials in humans would require approval from an Institutional Review Board (IRB) and the Taiwan FDA, as well as compliance with international regulations.
• Higher biosafety levels (BSL-2 or above) might be needed if mammalian or more complex systems are used.

These steps ensure that any future application of our engineered bacteria will be carefully tested and regulated before reaching patients.