Laboratory Safety
Safety in our project is not only about following laboratory protocols, but also about designing responsibly from the very beginning. Through Human Practices, we learned that certain cat allergen genes (e.g., Fel d 2) are linked to normal physiological functions in cats. This insight shaped our design choices: we deliberately limited Fel d 2 knockdown to in vitro control experiments only, avoiding any potential risk to animal health. By combining laboratory safety, biohazard management, and Human Practices feedback, our project establishes a continuous HP–Design–Safety cycle that minimizes risks to humans, animals, and the environment.
Laboratory Safety
Our experiments were conducted in a BSL-1 laboratory at Beijing Normal University. All members of the lab team received safety training before entering the laboratory, including the proper use of lab coats, gloves, and face masks. Hazardous chemicals were stored according to standard protocols, and all liquid and solid waste was disposed of in compliance with official regulations.
Materials Safety
We used Escherichia coli (lab strains) and yeast (Saccharomyces cerevisiae), which are widely used, non-pathogenic organisms. In addition, the shRNA sequences designed in our project target Fel d 1, Fel d 2, Fel d 4, and Fel d 7, which have no homologous sequences in human or other essential animal genes, ensuring no unintended effects on humans or other species.
Project Risk Analysis
Human Risk: Our RNAi constructs were carefully designed to avoid any sequence similarity with human or essential animal genes, preventing potential off-target effects.
Animal Risk (Cats): Although no in vivo experiments were conducted, we considered the safety of cats at the design stage. Through literature review and Human Practices interviews with veterinarians and experts, we learned that Fel d 2 is involved in normal physiological functions in cats. Therefore, we only used Fel d 2 sequences as laboratory controls in vitro and have no short-term plan to apply them in cats. This cautious approach helps ensure the biological safety of future applications.
Environmental Risk: No organisms were released into the environment. All genetically modified materials and microbial cultures were contained and safely disposed of.
HP–Design–Safety Feedback Loop
Our safety considerations were not limited to lab protocols but also shaped by Human Practices insights. During HP activities, veterinarians and experts emphasized that some cat allergen genes, such as Fel d 2, play roles in physiological health. This feedback directly influenced our design decisions:
We adjusted our RNAi targets to avoid applying Fel d 2 knockdown in vivo.
Fel d 2 constructs were kept as in vitro laboratory controls only, preventing potential harm to cats.
This demonstrates how Human Practices informed our design choices, which in turn strengthened our safety strategy, forming a continuous HP–Design–Safety cycle.
Risk Mitigation
a. Conducted rigorous off-target screening of RNAi sequences using NCBI BLAST to exclude similarity with human or other key animal genes.
b. Used standard plasmid vectors without high-risk antibiotic resistance combinations.
c. Applied strict microbial waste management: all microbial cultures, solid consumables, and liquid media were collected in biohazard containers, autoclaved at 121 °C, and treated with disinfectants (e.g., 10% bleach) before final disposal.
d. Chemical waste was stored and disposed of following institutional and governmental safety protocols.
Considered feline physiological safety: avoided direct use of RNAi constructs (such as Fel d 2) that could affect essential functions in cats, limiting them to in vitro assays only.
Ethics and Compliance
All work was restricted to the cell level and did not involve animal or human experimentation. Any future extension of the project to animal studies would strictly require institutional animal ethics approval. Furthermore, our project fully complies with both iGEM Safety and Security Policies and Beijing Normal University’s laboratory regulations.
Risk Table
| Risk Category | Potential Issue | Mitigation Strategy |
| Human | Potential off-target effects on human genes | Sequence design with BLAST screening to exclude human matches |
| Animal (Cats) | Potential interference with essential functions (e.g., Fel d 2) | HP-informed literature review; use only as in vitro control, no application in cats |
| Environment | Microbial waste leakage | Autoclaving cultures/consumables, chemical disinfection, safe waste disposal |
