Safety of project design

Our engineered chassis is E. coli Nissle 1917 (EcN) is a probiotics that has been safely used in clinical and commercial formulations for over a century. Its well-documented safety profile makes it an ideal host for therapeutic and diagnostic applications in the feline gastrointestinal tract. However, to further minimize risks, we implemented multiple strategies of biological and physical containment mechanisms.

First, to prevent survival after excretion, we implemented a temperature-sensitive suicide system (Figure 1). The feline intestinal environment maintains a temperature of approximately 38 °C, which is significantly higher than that of the external environment [1]. Our circuit utilizes two thermosensitive proteins, TEV ts-18 and TF ts-2, that inhibit each other under gut temperatures. However, once exposed to lower environmental temperatures, TEV ts-18 degrades TF ts-2, thereby releasing inhibition of the downstream endolysin gene. This triggers bacterial self-lysis outside the host, effectively preventing the survival in the environment and reducing the likelihood of gene transfer [2].

Figure 1. Cold-inducible switch design. Created by biorender.com.

At the hardware level, we incorporated an automated sterilization module into our intelligent litter box system to complement biological containment. The device is equipped with a UV-based disinfection unit that activates upon detection of fecal matter. Short-wavelength ultraviolet radiation disrupts bacterial DNA, inactivating any remaining engineered E. coli. The sterilized waste is then sealed in an enclosed compartment, minimizing the risk of microbial release, aerosol generation, or environmental contamination. This provides a secondary, physical layer of biosafety control.

To mitigate horizontal gene transfer (HGT) within the gut, all functional plasmids are chromosomally integrated into the EcN genome. This genomic integration prevents plasmid-mediated transfer via conjugation or transformation, ensuring that engineered genetic elements remain confined to the intended host strain.

Together, these strategies, temperature-sensitive self-lysis, hardware-based sterilizationand chromosomal integration stablish a multilayered biosafety framework that minimizes the ecological and genetic risks associated with deploying engineered probiotics.

Safety during experiments

During our project, all experiments were conducted in a BSL-1 laboratory using open bench spaces. Procedures involving bacteria were carried out in biosafety cabinets, and all team members wore lab coats and gloves throughout the entire process. We also remained aware of nearby butane burners to ensure safe handling of flammable materials.

Before commencing laboratory work, the team underwent comprehensive safety training, covering proper equipment usage, adherence to laboratory hygiene rules, and the appropriate disposal of hazardous substances. Supervisors provided detailed presentations for each experiment to address any potential biosafety concerns. All members received training on essential experimental procedures, including PCR, gel extraction, Golden Gate Assembly, transformation, and plasmid extraction. We were also trained on the handling, identification, and proper use of all laboratory equipment and materials, ensuring that every step of our experiments was conducted safely.

Certain experiments required the use of antibiotics to select for transformants. To minimize potential side effects to humans, our team conducted a survey before the project to ensure that none of the members were allergic to any antibiotics used. Antibiotics were stored and handled following appropriate security protocols. Other experiments necessitated the use of harmful reagents or procedures, and all team members strictly adhered to the guidelines provided by our lab manager and assistants during safety training. Similarly, hazardous chemicals such as Tris-HCl and methanol were stored in designated areas, and relevant procedures were conducted within chemical ventilation cabinets. All liquid waste was meticulously handled, ensuring the utmost adherence to safety.

To mitigate potential risks of gene leakage during wet lab processes, liquids and plates containing antibiotics were carefully placed in designated vessels and subsequently autoclaved for proper disposal. Discarded reagents and Petri dishes were placed into biohazard waste bags with clear warning signs and thoroughly taped to avoid possible exposure. Refrigerators and incubators were cleaned biweekly to maintain laboratory hygiene.

All agar artworks were created under BSL-1 laboratory conditions using E. coli BL21 strains. Only photographs of the finished artwork were taken outside the lab for exhibition purposes. No engineered or live bacterial strains were transported or displayed outside the laboratory, ensuring full compliance with biosafety regulations.

All procedures strictly followed established safety standards. Furthermore, all university laboratories in China undergo accreditation by the Office of the Ministry of Environment, confirming adherence to national biosafety protocols.

Contribution to Whitelist

In order to analyze the characteristics of keratinase, we hope to use cat hair as the substrate to test the enzyme's activity as part of our proof-of-concept. In accordance with the new rules of the high school competition, we carefully reviewed the whitelist and found the definition related to animal-related materials. Therefore, we politely asked the iGEM safety team via email whether we could use cat hair in the experiment.

We stated: "We plan to obtain the cat hair that has been professionally trimmed in veterinary clinics or beauty salons. These cat hairs were cut off for medical or hygiene purposes and then discarded." This is done to ensure the highest ethical and safety standards. For more details, please see the attached email below.

Our email has been replied to by the iGEM headquarters. Our request to use cat hair has been approved. At the same time, HQ replied that the part of the regulation for Whitelist and Check-in will change, and the logic will be consistent with the logic stated in our email. We are very grateful for HQ's reply and clarification, which demonstrates the importance of ethics and safety in this iGEM competition.

Future plans

In order to fulfill our group's final goal of marketing our project, the Clinical trails consent guidelines should be followed. A Clinical Trail Authorization(CTA) will be gained for clinical trails from Phase I to Phase III. Manufacturing permission for active pharmaceutical ingredients(AIPs) as well as our whole product would be obtained, following the Drug Admission Law(DAL).

References

↑ Levy, J. K., Nutt, K. R., & Tucker, S. J. (2015). Reference interval for rectal temperature in healthy confined adult cats. Journal of feline medicine and surgery, 17(11), 950–952. https://doi.org/10.1177/1098612X15582081

↑ Zheng, Y., Meng, F., Zhu, Z., Wei, W., Sun, Z., Chen, J., Yu, B., Lou, C., & Chen, G. Q. (2019). A tight cold-inducible switch built by coupling thermosensitive transcriptional and proteolytic regulatory parts. Nucleic acids research, 47(21), e137. https://doi.org/10.1093/nar/gkz785