Harmless Chassis

We have chosen E. coli BL21 as the biological chassis for our project. Prior to selecting our chassis organism, we conducted a thorough review of data from the China CDC, NIH Guidelines, and ATCC Product Sheet. This diligence ensures that our chosen bacteria fall within Risk Group 1 classification in our design. This classification signifies that our bacteria pose no threat of causing disease in healthy adults when handled in accordance with safe practices.

Trained Members

In addition, every team member has received professional laboratory training and strictly adheres to various laboratory safety protocols upon entering the lab. Each team member possesses good experimental skills, and we have dedicated laboratory managers who oversee safety within the laboratory. During experiments, our team members consistently wear lab coats and gloves and disinfect their hands before and after experiments to ensure their safety. Additionally, our laboratory equipment is routinely inspected by our team members and laboratory administrators at the end of each day, and we conduct ultraviolet sterilization of the lab to maintain a safe and sterile working environment.

Figure 1. Our team member in the lab

Safe Use of Reagents

To ensure safety, we thoroughly reviewed the MSDS for all chemicals, with special attention to formaldehyde and formic acid, which we are using in our experiments. This has equipped us with the necessary knowledge on how to respond in case of exposure. Meanwhile, when dealing with formaldehyde and formic acid, we definitely took protective measures.

Table 1. Main reagents used in our experiments

Additionally, the formaldehyde reagent was stored under sealed conditions at -20°C and divided into small portions for use. Formaldehyde was only used within a designated area with good ventilation, and all pipettes and other instruments in this area ware strictly used following proper safety rules.

In our lab, all biological waste was sent to designated university waste disposal services. We had essential safety features in place, including fire extinguishers, waste disposal units, first-aid kits, and a health center available 24/7 in case of emergencies. Our team ensured the presence of a lab technician to supervise our work at all times. We are immensely grateful to our lab technicians who assisted and supported us during our experiments.

Suicide Switch

To prevent any inadvertent release of our biological chassis into the environment, we've devised a distinctive regulatory pathway that incorporates a suicide switch controlled by an arabinose operon. In this system, the expression of the toxin MazF is under the control of the potent T7 promoter, while the expression of the antidote MazE is governed by the arabinose operon.

When L-arabinose is introduced into the culture medium, the modified bacteria commence the production of the antidote MazE. This antidote can form a complex with the constitutively expressed toxin MazF, rendering it non-lethal to the cells [1]. Consequently, in a culture medium supplemented with L-arabinose, the engineered bacteria can thrive normally.

Nevertheless, if these engineered bacteria escape into an environment lacking L-arabinose, they will perish due to the action of toxin MazF since they are unable to produce the antidote MazE. This ingenious design ensures that the engineered bacteria can only persist within a designated environment, thereby mitigating any potential repercussions on the external surroundings.

Figure 2. The genetic circuit of the suicide switch

Laboratory Safety Regulations

Our team rigorously adhered to a set of guidelines to ensure safety and hygiene within the laboratory. These guidelines mandated that all team members wear appropriate attire, including lab coats, masks, gloves, hairnets, and closed-toe shoes, at all times while conducting experiments. The rule of strictly prohibiting food and drinks in the lab was enforced without exceptions.

Figure 3. The laboratory safety code

While the majority of the components used in our project were non-hazardous and safe, we took special precautions when handling the potentially hazardous reagents like formaldehyde, including storage it in a designated area. After use, we ensured responsible disposal of pipette tips, gels, and the gloves worn during the experiments. These practices not only safeguarded the well-being of all team members but also preserved the integrity of our work environment.

References

[1] Yamaguchi, Y., & Inouye, M. (2011). Regulation of growth and death in Escherichia coli by toxin–antitoxin systems. Nature Reviews Microbiology, 9(11), 779–790.