Safety has been attached great importance in every year's project and experiment for HZAU-China. This year, our team is acutely aware of the criticality of safety and security for both researchers and the environment. Not only does safety shield individuals from potential harm, but it also safeguards against the inadvertent release of pathogens.

We stringently adhere to the safety regulations and policies mandated by the iGEM authority, and meticulously consider every potential safety issue associated with our project. It‘s necessary to think about each aspect and give corresponding solutions as pre-arranged planning to avoid uncertainty, because our products are intended to be put into practical production through cooperation with relevant companies and governments.

At the beginning of the project, researcher Xia Chunjiao provided us with adequate experimental safety training. She introduced us to all the risk factors in the lab and taught us how to perform the experiments correctly, which laid a solid foundation for our safety. Figure 1. Experimental safety training.
Safety training enables us to understand the laboratory environment and ensure our own safety.

Adequate education is a prerequisite for safety. Professor Qi Yingchun, who is in charge of biological laboratory security, conducts offline laboratory safety training for the iGEM project team members every year and requires them to pass corresponding online laboratory safety exams (Figure 2). Each year, the existing members of our laboratory also guide new members in conducting safe laboratory operations. Figure 2. Laboratory safety examination certification of iGEMers.
This examination ensure that all team members strictly comply with the safety standards of the laboratory.

In the middle period of the project and when the experiment was busy, Professor Qi Xinchun once again held a safety training meeting for us, emphasizing the importance of experiment safety and introducing many precautions in operation, so as to ensure the safety of all personnel as much as possible. In short, safety education runs through the whole process of our project. Figure 3. Safety training meeting.
The safety meeting once again reminded us of the importance of safety to ensure the smooth conduct of the experiment.

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, the E.coli and B.subtilis are our chassis organisms. The E.coli DH5α, BL21 (DE3) and B.subtilis BS168 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 plant to the external environment, we used a photosensitive suicide device. More details are shown below.

Safety of chassis and parts

Frist, The biological chassis of our project is B.subtilis and Bacillus Velez, which is widely recognized as a probiotic with no known harm to human health or the environment. All of the parts used in our project will only be used to kill nematodes and commit suicide of engineered bacteria, and will not cause any harm to humans, animals or plants.

Safety in design

Secend, considering the leakage of engineered bacteria, We used KillerRed, a phototoxic protein, to execute suicide function. According to research, KillerRed is a red fluorescent protein that produces reactive oxygen species (ROS) under yellow-green light (540-585 nm). According to its characteristics, our engineered bacteria can be killed under both artificial green light conditions and natural daylight conditions to ensure its full safety, which has been verified in the experiment and shown in the results. Figure 4. The gene circuit design and protein functions of safety module.
The KillerRed protein produces ROS when exposed to light, killing the engineered bacteria.

Safety of implementation

Last, considering the practical application of the project, our chassis biology chose to use two probiotics that are symbiotic with plants in the natural environment - Bacillus subtilis and Bacillus velezensis, which have a certain control effect on pests. Based on our fieldwork at HP, we learned that these two bacteria are often made into powder for practical application in the field. Therefore, our plan is to directly process the engineered bacteria into powder, which is very conducive to actual production and use.

Safety of laboratory

Our lab locates in the main hall of College of Life Science and Technology, Huazhong Agricultural University. The main hall has an access control system (Figure 5), which can identify the student’s ID and only allow authorized students to enter. Meanwhile, we have set up a lab duty schedule to ensure that there is always a responsible student on duty in the lab every day. The teacher in charge of safety management for our college also provides regular safety training for each team member and conducts regular safety inspections of the lab. Figure 5. Our entrance guard system.
Only team members have access to our laboratory.

Our laboratory is equipped with the fume hood, medicine cabinet (Figure 6) and all the biological waste generated during the experiments is managed by trained stuff. At the same time, we have established strict zoning rules and laboratory waste disposal requirements. So all of this measures can ensure that we can do experiment in a safe environment. Figure 6. The safety of our laboratory.
A.The overview of our laboratory. B.Fume hood. C.Medicine cabinet. D.Trash bin for biological waste.

Safety of experiment

In our project, we mainly use Escherichia coli and Bacillus subtilis as engineered bacteria that pose extremely low health risks to humans and animals and can be operated in BSL-1 laboratories. In the experiment, we will also use plants as the experimental materials for our final effect, which are all germinated from seeds in the laboratory, and also do not enter the external environment.All these can ensure that our engineered bacteria do not leak into the natural environment.

When conducting experiment, all researchers wear gloves, masks, and lab coats (Figure 7). 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. Figure 7. A researcher operated with bacteria.
All experimental operations will strictly follow safety specifications.

We are also very careful in the treatment of experimental materials to avoid the occurrence of dangers. In the transmission electron microscope experiment, uranium acetate and other dangerous substances are carried out by professional operator Zhang Yuan, to ensure the safety of personnel.

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.