Safety

Lab Safety

Our laboratory operates under BSL 1 conditions, primarily handling T7 bacteriophages and the E. coli MG1655 strain. All laboratory members are equipped with necessary Personal Protective Equipment (PPE), including lab coats, face masks, and gloves. We adhere to the division of aseptic operation areas, with particular attention to the separation between rest areas and laboratory spaces. Additionally, in the electrophoresis area, extra caution is exercised to avoid the potential hazards of direct contact between gel dyes and the human body. We have implemented a comprehensive safety training program. Prior to entering the laboratory, all team members receive mandatory laboratory safety training from our Principal Investigator (PI) and consultants. This training includes biosafety training, professional training, and emergency handling training. During the experiment process, all experiments are conducted in biosafety equipment that meets experimental requirements, such as biosafety cabinets and autoclaves. We also perform regular maintenance and inspections on the experimental equipment to ensure it is in good condition. There are clear standard operating procedures for laboratory safety, and all experiments are carried out in accordance with the requirements. An emergency response plan for laboratory biosafety incidents has been developed, which enables effective response to unexpected events and clarifies the division of responsibilities. This equips our team with the ability to follow emergency response protocols and operate various laboratory equipment safely. For hazardous equipment, we have affixed instructions and preventive warnings to reduce potential accidents. Furthermore, we have established a strict waste management system to prevent contamination and infection. For disposable and reusable bacterial culture containers, we follow a cyclic handling process, which includes proper disinfection, cleaning, and reuse.

Basic Laboratory Training

Before starting to clone our fragments, we had a full academic year dedicated to laboratory training to consolidate our lab skills. We learned how to conduct molecular biology experiments, such as performing agarose gel electrophoresis, PCR, and pipetting. Meanwhile, all of us studied relevant biosafety knowledge, enabling us to create a proper environment for storing, transferring, and using our samples. We gained an understanding of different biosafety levels, what to do in case of chemical spills, and how to address other potential issues in the laboratory. Through such learning and management, our team has effectively emphasized biosafety and its importance.

Safety Research Design

The bacteria we designed can produce the protein capsid of T7 bacteriophage and package the plasmid containing the toxin gene into the capsid. When the phage-like particles (PLPs) adsorb to the surface of pathogenic bacteria, the plasmid will be injected into the pathogenic bacteria, which can be used to kill bacteria harmful to crops. We have also modified the tail fibers of the phage-like particles to enable them to specifically target different pathogenic bacteria. Our work will help eliminate bacterial diseases in crops (cash crops), achieve significant yield increases, and address potential hunger and nutrition issues. We use E. coli MG1655 to produce various phage-like particles that target and kill pathogenic bacteria, and this strain is classified as Risk Group 1. In the project design, we have developed a toxin-antitoxin system to prevent the escape of engineered bacteria during fermentation. An arabinose-inducible promoter (PBAD) is used to drive the expression of the antitoxin gene mazF. During fermentation, arabinose is added to the culture medium to induce the chassis bacteria to express the antitoxin, ensuring their normal survival. If the engineered bacteria escape into the open environment, the antitoxin gene mazF in their cells cannot be expressed, causing the engineered bacteria to be killed by the toxin produced by the toxin gene mazE on the toxin-containing plasmid pUC19-pac. This suicide switch ensures that any engineered bacteria escaping during fermentation cannot survive in the external environment. During product application, our chassis bacteria are completely lysed by ultrasonication to release the phage-like particles they synthesized. This physical sterilization method ensures that there are no viable chassis bacteria present when the product is applied. These two approaches effectively prevent the leakage of chassis bacteria and significantly enhance biosafety. Finally, bacteriophages exhibit high specificity, with a very limited host range they target. We have listed most of these host ranges below; the narrow host range indicates that the probability of them posing a toxic risk is extremely low. Our work only involves the small-scale production of T7 bacteriophages, which will not cause any environmental issues. Throughout our entire xperimental process, we strictly adhere to biosafety protocols and conduct experiments under the supervision of our instructor.

Biosafety and Synthetic Biology Discussion

In our interviews with professors, organizations, and experts, we discussed biosafety issues to gain their insights, opinions, and potential concerns. We were particularly interested in the prospects and future of using synthetic biology-based phage-like particle packaging to address pathogenic bacteria. During our interviews, we asked questions about biosafety and engaged in discussions with the invited interviewees. When interviewing Professor Dong Wubei, he advised us to fully consider the potential transferability of toxin-containing plasmids in E. coli. Professor Zhou Feng mentioned that existing frameworks struggle to dynamically assess the long-term behavior of engineered microorganisms in complex ecosystems. In response to these suggestions, we designed our product to be phages obtained after lysis, and used ultrasonication to thoroughly inactivate bacteria, preventing the escape of chassis bacteria. Several scholars unanimously emphasized the importance of the “precautionary principle” and “ecological adaptability” in terms of biosafety. Inspired by their insights, we designed our product application method around the concept of “prevention is better than treatment”, aiming to conduct preventive application before the high-incidence period of diseases to reduce environmental impact. We have received a variety of responses and gained insights into their views on synthetic biology. Some hold reserved opinions and are concerned about the challenges posed by synthetic biology, while others look forward to the breakthroughs it can bring to humanity. We are aware that biosafety is the top concern for many people.

In the project, we adopt a serious and rigorous attitude towards safety. Safety is not an additional afterthought for remediation, but an element that runs through the entire process. Additionally, we recognize that innovation and safety have never been an either-or choice. Technological progress can only move forward steadily and achieve long-term development when it is on a safe track. This is not only our response to current concerns, but also our commitment to sustainable development.