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Our project aims to leverage engineered E.coli to produce cost-effective sugar substitute, tagatose , for use by factories and companies engaged in the production of food and beverages. In this context, ensuring the safety of our project has been prioritized as a primary concern. Throughout the project, our team collaborates to identify and manage all potential risks, while minimizing hazards that may affect our team members. Under the supervision of our instructor,Xiao Xiao, and our principal investigator and advisors, including Liu Peipei and Zhang Xiaoyu. This document will outline the safety measures implemented across all aspects of our work.

Escherichia coli strain DH5a and BW25113 were used in this project. Any other organisms will not be used in our project, and none of our parts could be hazardous either on their own or in the context of our project. Our engineered organisms or parts are also unable to spread in the environment. Because after the completion of fermentation, the supernatant is collected by centrifuge, and the E.coli were autoclaved. All the broth used in our project were autoclaved after use. Then the containers used were further sterilized use 84 disinfectant.
We are using GelRed, a kind of DNA gel stain, resorcinol (Toxic if swallowed) and alcohol (highly flammable chemicals) in our lab, which may cause certain safety or environmental hazards. GelRed was used for electrophoresis, which was thought to be harmful for human health, even though its instruction said it is safe in animal test. Resorcinol was used for tagatose determination, we used it at a very low concentration (≈0.2%) which is 15 times lower than its toxic dose. And alcohol was used for alcohol burner, which is also essential for sterile operation in clean bench. For GelRed, we isolate it to the contaminated area. We have to wear a pair of plastic gloves over the rubber gloves when using the stain. For resorcinol and alcohol we carefully use them and under the supervision of our instructor.
We are not planning to do any prohibited activities or activities that require advance permission, ensuring utmost safety. We intend to use E.coli as a cell factory to produce tagatose. We are interested in PGP (NC_0 1 374 1.1), Gatz (WP_0 14433578 ) over-expressing in E.coli, we have found the sequence for the proteins in some publications and have them synthesized by a commercial supplier.

Our lab is graded level 1 in terms of biosafety, so all the materials we used impose little or no risk on individuals' health. Our lab is strictly divided into three areas, working area, resting area and lab area. Lab area is further separated into contaminated and non-contaminated areas. Agarose gel electrophoresis can only be conducted in the contaminated area with special pipettes and conical flasks that are only for the use in contaminated area. All the solutions containing microorganisms are collected in a specific container first and then added with 84 disinfectant (bleach) before being discharged. We also sterilise peri dishes (plates) and all other contaminated materials using high temperature and high pressure environment before disposing them therefore make sure that no living microorganisms are released from our lab.
We wear gloves, lab coats and tie up our hair when entering the lab area in order to protect ourselves. Before we enter the lab and start to do experiments independently, all of our members have to be trained for correctly and safely using the lab and all of us have to pass an exam about the lab safety to start taking part of our experiments. Besides, we have a set of rules including not to eat or drink in lab, not to touch phone in lab, not to bring laboratory waste out or in the wrong bin, etc. There is a surveillance system above the lab area to make sure we are always under supervision.
Accident reporting (system to record any lab accidents) Personal Protective Equipment / PPE (wearing lab coats, gloves, eye protection, etc.) Inventory controls (tracking who has what physical materials and where the materials are) Physical access controls (controlling who can access your lab or storage spaces) Data access controls (controlling who can access computers or databases) Lone Worker or Out of Hours policy (procedures for working alone or at times when normal support is unavailable) Medical surveillance (finding out if you get sick because of an organism or chemical you used) Waste management system (such as decontaminating waste before it leaves your institution)

We are collecting some data about people, such as their opinions, quotations, medical history, gender, behavior, attitudes, or concerns. We have obtained formal / institutional approval for our work (or will obtain it before starting data collection). We have confirmed that relevant laws, regulations, and institutional rules do not require us to get formal approval.

In summary, our team integrated biosafety in three ways throughout every detail of the project, including biosafety, lab safety and human practice safety. We strictly ensure the safe deployment of engineered modified bacteria in real-world environments. In addition, our HP education activities impact biosafety to a wider audience with different ages. We also take steps to ensure the safety of everyone at work. The different safety measures ensured that our project ran successfully and could be an example for future iGEM teams.