Introduction
Our team sets safety as an integral part of science. To eliminate the safety concerns, we viewed the safety problems from three different perspectives — the “Project safety” module, the “Lab safety” module, and the “Future plan” module.
The “Project safety” Module
In our project, the genetically engineered bacteria are put in the capsules and target the human lumen. The cold-inducible kill switch is designed in our plasmid to ensure biosafety and eliminate the potential release of genetically engineered E. coli. Specifically, this cold-inducible kill switch is susceptible to rapid environmental temperature changes. The temperature is approximately 37 degrees Celsius in the human lumen, and any sudden exposure to lower temperatures in the in vitro environment will trigger the switch. The switch will respond swiftly to temperature variation, which relies on the TEV ts-18 and TF ts-2 proteins, which have corresponding temperature-transition points at 36.5 degrees Celsius. In this system, TFts and TEVts mutually inhibit each other, and the TFts-repressed PR promoter regulates the expression of TEVts. At lower temperatures, the constitutively expressed TFts are cleaved and inactive by TEVts. Consequently, the cold-inactivated TFts lose their ability to tightly repress the downstream suicide endolysin and TEVts. This allows TEVts to further cleave TFTs, leading to the expression of the suicide endolysin and ultimately triggering the self-lysis of E. coli.
Figure 1. The cold-inducible circuit design
To enhance the suitability of our therapeutic bacteria for medical applications, we have devised a strategy involving the preparation of freeze-dried powder and its encapsulation within an enteric capsule shell. This shell, being resistant to gastric acid and soluble in the small intestine, serves as a protective barrier, shielding the therapeutic bacteria from destruction. Upon detection of the target pathogen, S.aureus, by our engineered bacteria through the QS molecule pathway, the release of antibacterial peptides and endolysin is triggered to inhibit the growth of endolysin. The tight promoter precisely regulates the release of the endolysin, ensuring efficient and controlled action, leading to the destruction of E. coli.
The “Lab safety” Module
To ensure compliance with established standards, all university laboratories in China undergo accreditation by the Office of the Ministry of Environment. For our project this year, we conducted our experiments within a BSL-2 laboratory, with team members consistently wearing lab coats and gloves. Experiments involving bacteria were conducted within the biosafety cabinet while remaining cautious of the nearby butane burners.
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. Additionally, our supervisors provided detailed presentations on each experiment to address any biosafety concerns that may arise.
Given that our project involved the use of antibiotic resistance genes, we implemented special precautions 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.
Certain 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 the experiment safety training.
To sterilize the biosafety cabinets and the equipment housed within, UV light was utilized. Measures were taken to prevent direct exposure of individuals to UV light. When visualizing agarose gel, we employed the gel imaging system in conjunction with standard laboratory procedures involving a blue light transilluminator.
In the process of selecting transformants, antibiotics were utilized, which can potentially pose harm to sensitive individuals. Therefore, we gathered information regarding any allergies among team members. Appropriate storage and handling of antibiotics were implemented, following strict safety measures in accordance with disposal protocols.
Other hazardous chemicals, such as Tris-HCl and methyl alcohol, were stored in designated areas, and relevant procedures were conducted within chemical ventilation cabinets. Liquid waste was meticulously handled, ensuring the utmost adherence to safety measures.
The “Future plan” Module
For our project, we would need to comply with the Clinical trials consent guidelines and obtain a Clinical Trial Authorisation (CTA) from the National Medical Products Administration (NMPA) to conduct Phase I to III clinical trials. Additionally, under the Drug Administration Law (DAL), we would need to obtain manufacturing permits for both the finished pharmaceutical products and the active pharmaceutical ingredients (AIPs) involved in our treatment approach.