Team:USTC

Overview

    Given that safety played a pivotal role in lab, we adopted some strategies to ensure the safety while performing our experiments. We took biosafety into account when we designed our project. Our lab’s safety regulations and national guidelines we followed are mentioned bellow.

Project

    All materials we used in the project were strictly selected, following the P1 laboratory regulations and were harmless to humans or the environment.

    In general, our project was conducted at a very low risk because no pathogenic bacteria or dangerous chemical reagents were used during the experiment. As for the implementation of our project in the real world, the product will meet the requirements of security issues.

    Our strain, Yarrowia Lipolytica, is “generally regarded as safe” (GRAS) organism，whichcan be used in the food and nutraceutical industry (USEPA ). So even there is an accidental leakage problem in the course of experiment, it will not lead to very serious consequences.

    In addition, we have also carried out an experimental design to increase the safety of the design. For Y.lipolytica, RAS1 and RAS2 are essential for their survival, and the yeast will die when both of them are inactivated. Therefore, we combined two molecular switches to design a “suicide switch” that works under certain condition. In an environment rich in Cu²⁺, the copper-binding transcription factor CRF1 initiates the expression of promotor P_MT-1 and that activates the transcription of the Cas9 gene; when there is blue light in the environment, the blue light induced activator initiates the transcription of RAS1 and RAS2 gRNA. After binding to gRNA, Cas9 knocks out RAS1 and RAS2 genes respectively, causing yeast death. Compared with the use of a single transcription factor to construct a suicide switch, our design separates the two key components and makes the switch not falsely open due to accidental factors. In this way, we can not only further ensure safety, but also guarantee the continued production of borneol.

Figure 1. The design of the “suicide switch”. When blue light and Cu2+ exist, the cell will die.

Lab

    Everyone in our lab received comprehensive safety training before we started the experiment. For example, we must wear experimental gloves and safety goggles correctly when doing experiments. In addition, all of us are familiar with the relevant safety regulations and adhere to them under the supervision of our consultants and PI.

    The reagents and instruments we use are correctly labelled and placed according to the guidelines.

    Upon completion of the experiment, we will properly handle chemical and biological waste to avoid potential laboratory risks.

Guidelines

    All laboratory experiments we conduct are subject to the General Guideline for Biosafety in Microbiological and Biomedical Laboratories ( Chinese Center for Disease Control and Prevention ). In addition, we also stipulate the safety regulations that our laboratory must comply with (Figure 2). These include, but are not limited to, the following:

    (1) Responsible individuals

    (2) Fire safety

    (3) Security regulations of electricity

    (4) Safety rules of hazardous chemical reagents, ranging from the purchase to storage

    (5) Correct disposal of chemical and biological wastes

    (6) Biological safety

Figure 2. The management regulations of our laboratory