Safety has been attached great importance in every year's project and experiment for HZAU-China. This year, our team recognizes the importance of safety and security to researchers and the environment. Safety not only protects people from any harms but also prevents the accidental release of pathogens and toxins. We not only abide by the safety rules and policies provided by the iGEM authority strictly, but also take every possible safety problem of our project into our consideration. As our product will be applied in actual working conditions, it is necessary to consider every aspect and provide corresponding solutions as part of pre-arranged planning to avoid uncertainty.

Our lab locates in the main hall of College of Life Science and Technology, Huazhong Agricultural University. The main hall is equipped with an access control system, which can identify the student’s ID and only allow authorized students to enter (Figure 1). The teacher in charge of safety management at our college provides safety training for each team member and routinely inspects our lab for safety compliance.

Professor Qi Yingchun is responsible for the safety and security of the experimental building. She worked as a security guard at Huazhong Agricultural University for decades, and taught biological experiments in the university. Every year, before the iGEM project officially starts the experiment, each team member must attend the safety education meeting held by Professor Qi and Professor Xia and pass the corresponding online laboratory safety examination.

Our laboratory is equipped with the fume hood, medicine cabinet (Figure 3). In addition, all the biological waste generated during the experiments is managed by trained stuff. So all of this measures can ensure that we can do experiment in a safe environment.

All researchers wear gloves, masks, and lab coats (Figure 4). When handling volatile or irritating items, the experimenter is required to wear goggles to ensure safety. For using the instrument, the researchers strictly followed the instruction protocol to operate.

In our project, we need to use human IgE samples to evaluate the efficacy of scFvs produced by engineered bacteria in blocking the interaction between Fel d 1 antigen and human IgE. So we need to apply for medical ethics through the Ethics Committee of Huazhong Agricultural University. After the official approval of our experimental plan, we will recruit 5 volunteers with a history of allergic reactions to cat in September (Ethical numberID Number: 202309070014).

The collection of blood samples will be carried out by professional operators from the laboratory department of the university hospital, and the collected serum samples will be stored at -20℃. We promise to only use these serum samples in the laboratory environment.

At the same time, we also used cat saliva and teeth in our project to evaluate the effectiveness of bacterial colonization. Cat saliva and teeth come from Pet Kang Animal Hospital, located very close to our school. Veterinarians obtained 2-4 milliliters of saliva samples during sterilization surgery on a cat with stomatitis.

Moreover, we had submitted the Check-in-form and got a permission. None of this would violate Chinese biosecurity policy.

Safety has always been the focus of our project. We have completed and submitted a safety form that includes detailed information about our project. In our project, only the E. coli as our chassis organisms. The E.coli DH5α, BL21(DE3) were used to implement the experience of gene clone and expression. At the project design level, in order to prevent the leakage of engineered bacteria from the cat's mouth to the external environment and the cat's own digestive tract, we designed a temperature and pH sensitive suicide circuit. More details are shown below.

Safety in project design

The chassis of our project is E. coli Nissle 1917, which is widely recognized as a probiotic with no known harm to human health or the environment. Then, considering the implementation of our bacteria, We designed two suicide schemes. In the first scheme, the expression of RelE is inhibited by the heat suppressed RNA thermometer in the cat's oral cavity, and it can continue to be expressed in vitro and cause bacterial suicide. In another scheme, engineered bacteria will continue to express HepT toxin and MntA antitoxin at oral pH, and the bacteria will not die; When bacteria entering the digestive tract, a decrease in pH leads to the activation of the acid inducible promoter asr to express CI protein, which inhibits the expression of MntA and thus performs suicide function.

Safety of implementation

In specific experiments, we used Escherichia coli DH5α, BL21(DE3) is used for gene cloning and expression, but these strains are usually not used in daily products. Considering the practical application of this project, our chassis has chosen to use a beneficial probiotic for cat health - Streptococcus salivarius K12, which can to some extent inhibit the pathogens of bad breath and dental caries. Based on HP's research, we have learned that cats are more inclined to lick rather than bite. Therefore, our plan is to incorporate engineered bacteria into the nutritional cream. As cats eat, the engineered bacteria will stay and colonize in their mouths. At the same time, nutritional creams can also provide some nutrients for cats.

In compliance with iGEM's safety policies

Our project does not involve animal use, human experience, gene drive, coronavirus or the use of modified organisms, as well as any part of the WHO list containing sequences related to antimicrobial resistance. The research of human practice is divided into investigation, interview, teaching and teaching activities. All projects are pre approved by the review committee of our school and conducted under the good guidance of our PI and lecturers. As for the "Do not Release Policy", we strictly abide by the relevant rules of the laboratory to prevent engineering bacteria from escaping from the laboratory.

In compliance with iGEM competition safety rules

On the basis of following the security policy, we also strictly abide by the security rules of iGEM. We have filled in the safety form before the deadline. In addition, as mentioned earlier, our project does not involve animal use. All experiments carried out in our team's laboratories are always carried out in strict accordance with the principle of safety.