Our project aims to use engineered yeast (S. cerevisiae) to produce economical fragrace fixatives such as ambrein, santalol and bisabolene for factories and companies producing perfumes or other fragrance products. Under this circumstance, guaranteeing the safety of our project has been prioritized as one of the main concerns. During our process, LINKS-China makes joint efforts to identify and manage all the risks and minimize hazards that may pose to our members, under the guidance of a commercial team instructed by Shenzhen LINK SPIDER Co. Ltd., Jiawen Chen, our instructer, and our PI and advisors (including Boxiang Wang, Sha Zhou, Zhengyu He...). This page will discuss the safety measurements we took in every aspect.

Whole Organisms Used

Escherichia coli (E. Coli)-strain DH5a Saccharomyces cerevisiae (Yeast)-strain CEN. PK2-1C 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 centrifugation, and the yeasts are removed. The target product will be obtained after a series of process treatments. For the yeasts, we will use 84 disinfectant for sterilization and then discard them.

Chemicals Used

We are using SYBR DNA gel stain (carcinogen), Dodecane and hexane (highly flammable chemicals) in our lab, which may cause certain safety or environmental hazards. Specifically, SYBR dna gel stain is essential for electrophoresis, and dodecane and hexane are vital to extract Santalol, Ambrein and Bisabolene. However, for SYBR dna gel stain we isolate it to the contaminated area(explained in labotary safety). We have to wear a pair of plastic gloves over the rubber gloves when using the stain. For dodecane and hexane we carefully use them in places away from fire source and under the supervision of our instructor.

Experiments Done

We are not planning to do any prohibitied activities or activities that require advance permission, ensuring utmost safety. We intend to use S. cerevisiae as a cell factory to produce santalol, ambrein, and bisabolene. We are interested in tHMG1(BBa_K412019), IDI1(BBa_K4121025), ERG20_F96W(BBa_K4274002) overexpressing in yeast, which would then be extracted from the yeast genome and otherwise synthesized by a commercial supplier. In addition, a type of cyclase from B. megaterium, BmeTC and its variants (BBa_K480500-BBa_K4805003) and santalene synthase from C. lansium, CYP736A167(BBa_4805008), and SaCPR2(BBa_4805009) from S. album would be heterologously expressed in yeast. We have found the sequence for the proteins in some publications and have them synthesized by a commercial supplier.

Laboratory Management

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 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.

Personal Protection

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 sefety to start taking part of our experiements. 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.

Biosecurity Measures Used

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)

Surveys, Interviews, Human Subjects Researches

We are collecting any 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

Legislative proposal

Our project is closely related to biosafety. We believe we have the responsibility to prevent microorganisms releasing without being treated safely and we are making efforts in it throughout our process and thus provide a safer platform for doing experiments with engineered strains. Through reviewing related data and closely studying the Tianjin Biosecurity Guideline for Codes of Conduct for Scientists developed by Tianjin University, we learned and applied the national biosafety governance throughout the whole experiment. We also strictly follow Link Spider Laboratory Experimental Safety Rules and Regulations, which is shown below.

Do not Release Policy

We invite people to tour our lab but they are restricted from taking anything away with them. Visitors must wear labcoats, gloves and masks when entering the lab. Gloves and masks are spraved by concentrated ethanol before beingdisposed into bins,. Lab coats are reusable but they are sterilised with high temperature first. For our education and entrepreneurship this year, we mostly use videos and pictures to present our projects so no contaminated products are taken away.

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 the LINKS-China project ran successfully and could be an example for future iGEM teams.

灵蛛实验室实验安全规章制度. (n.d.). Weixin Official Accounts Platform. Retrieved October 9, 2023, from https://mp.weixin.qq.com/s/pQ43f0CAGvLCzbkJHh2_bQ