Before starting the project, we first analyzed the potential risks we might encounter. We eventually came to the conclusion that our risks mainly come from three parts:

1. the organism we use and its metabolites;
2. laboratory activities;
3. risks in the implementation process.

This year, we choose a very normal organism in the environment, E. coli, as our chassis. According to the registration form, E. coli can be classified as a risk level 1 organism, which means it has a relatively low risk of affecting our health. Therefore, we assessed the overall risk as low. However, in order to reduce horizontal gene transfer and avoid leakage, we still found it necessary to design an off switch for our engineered bacteria. Of course, our laboratory activities may bring a certain degree of safety risk, which we must try to avoid as much as possible. In addition, if our project is able to develop to practical use, then the harm that the bacteria and their residues may cause to humans and the environment after the dyeing is not negligible.

To ensure biosafety, it is necessary to prevent our engineered bacteria from posing a threat to the surrounding environment. Because our bacterial system is light-regulated during staining, we normally store it in the dark. So we chose yellow-green light, which is present in normal light but not in the light control system, to trigger suicide. We chose KillerRed that had been used in the iGEM Parts (BBa_K4628047) to commit suicide. According to research, KillerRed is a red fluorescent protein that produces reactive oxygen species (ROS) under yellow-green light (540-585 nm) and can be used as a kill switch for biosafety applications.

To achieve the suicide function, we introduce lacuv5 promoter that regulates the expression of the KillerRed module. Once the engineered bacteria are released into the natural environment, they will activate their suicide mechanism under the induction of natural light in the environment.

To test whether the killerred system is still applicable in E. coli, we did a simple and interesting light-induced experiment. Through light experiments, we showed that the viability of the strains carrying phototoxic proteins was significantly reduced after white light exposure on the plate.

In order to reinforce the management of the labs, the official of our university delivered management regulations. All of our activities are instructed under the official guidance. Besides, our lab also special regulations on management of instruments and equipment, which we must obey during experiments.

To gain access to our lab, we must apply on the lab website and take a test. The test included basic experiment skills, lab cleaning, experimental waste disposal, ethics, chemical reagents storage, lab rules, equipment safety, and emergency measures. Only those who pass the test are allowed to carry out experiments in our lab.

Associate Professor Yang Fan is the instructor of our lab. She has been in charge of our lab for 8 years. She is familiar with most molecule cloning experiments and can offer suggestions at any time. So our experiment specification is under the control of the instructor in order to avoid any possible danger. Before we began our business in the lab, we also had a 'safety education class' delivered by her to train us the specific rules to follow in the lab and how to deal with the possible emergency. When we are carrying out our experiments, our instructor will supervise our experimental operations, decreasing the posibility of unnecessary danger and damages.

Our laboratory has a dedicated lab instructor, who trains us on laboratory procedures, reviews our experimental plans, and ensures compliance with laboratory safety requirements. In addition, our team members are vigilant about the general biosafety standards of synthetic biology, consult literature to design more efficient safety modules, and regularly communicate with our mentor to discuss and optimize product safety.

Our work is carried out in strict accordance with the relevant laws and regulations and campus safety rules.

Ministry of Education of the People's Republic of China has released the "Safety Regulations for Higher Education Laboratory" and distributed it to all higher education institutions.

At Zhejiang University, the Laboratory Equipment and Management Department is responsible for the supervision and safety management of all laboratories.

For biosafety considerations, in order to prevent engineered bacteria and their related metabolites from causing harm to the environment or human beings after fabric dyeing, we also designed a hardware biosafety module.

The hardware biosafety module is derived from the hardware-based temperature control module. According to the experiment, all the engineered bacteria would lose their biological activity in 10 minutes at 70℃. Therefore, we controlled the temperature control system and heated the engineered bacteria after dyeing, so that the engineered bacteria could maintain for 10 minutes at 75℃.

Associate Professor Yang Fan is the instructor of our lab. She has been in charge of our lab for 8 years. She is familiar with most molecule cloning experiments and can offer suggestions at any time. So our experiment specification is under the control of the instructor in order to avoid any possible danger. Before we began our business in the lab, we also had a 'safety education class' delivered by her to train us the specific rules to follow in the lab and how to deal with the possible emergency. When we are carrying out our experiments, our instructor will supervise our experimental operations, decreasing the posibility of unnecessary danger and damages.

Before the engineered bacteria are officially released into the environment, rigorous biosafety test in accordance with the requirements of national governments and relevant departments will be conducted. This is to ensure that the engineered bacteria and related biological components will not cause harm to the environment. Our team will also work under the guidance of technical personnel to modify the biological components and optimize them to be harmless to the environment, thus minimizing any potential risks. Complete and reliable experimental data will be included in the preparation of the application documents for approval, thus providing the basis for patent application and application approval.