iZJU-China

Lab safety

Safety Training

iZJU-China is committed to establishing a safe environment for experiments and research. As arranged by the institute, we participated in Laboratory Safety Training at the beginning of our freshman year. The presentation file used in the lecture can be downloaded here. After the lecture, we took a Safety Test and each team member has passed it in one trial.

Before our experiments started, we reviewed the Laboratory Safety Manual again, which can be downloaded here. Below is a picture of team members reviewing the security manual. Advisors Kexin Nie and Jingyi Tian also provided us some detailed and specific guidance.

Experimental Safety

Personal protective equipment is mandatory for daily laboratory work. When we enter the lab, we wear white coats and nitrile gloves. When necessary (for example, when handling hazardous chemicals), we wear safety goggles. Since the experiment was conducted mainly during the summer vacation, we specifically reminded members to wear closed toe shoes and long pants when entering the laboratory. We also specifically remind our female team members to tie up their long hair.

The safety of the team members during the experiment has been effectively guaranteed. has been effectively guaranteed. The laboratory is BSL-1. Experiments involving harmful chemicals were carried out in the fume hood, including the preparation of gels in SDS-PAGE, the addition of DNA loading buffers, and experiments related to α-pinene and α-pinene oxide.

We focused on biosafety. All of our experiments used non-pathogenic strains of E. coli including BL21 and DH5α (Risk Group 1). During the experiments, disposable experimental consumables exposed to bacteria were discarded as medical waste. The waste liquid of cell culture was treated with 70% alcohol and then dumped into the sewer. Vessels such as flasks are bleached and then autoclaved. Experiments related to bacteria were conducted in the clean bench, which was cleaned with 70% alcohol and then illuminated by UV light. These prevent our GMOs from being released into the environment and cross-contamination.

Figure 1. Summary of lab safety

Project Safety

Project Design

The design of our project is based on the principle of security. We hope to achieve an efficient degradation of α-pinene in a way that is free from hazards.

The bacterium we used for genetical engineering is E. coli and the exact strain is BL21 Star (DE3). BL21 Star (DE3) is a derivative of BL21, which is the most common example of E. coli B strains and have been widely used in research and industry. Since E. coli has the advantage of whole genome sequencing with a mature gene cloning expression system, it is approved by the Food and Drug Administration (FDA) as a safe gene engineering receptor organism (1).

The parts we designed are supported by literature. In the conversion of α-pinene to α-pinene oxide, we intended to introduce pCDFDUET-1 p450bm-3qm glcdh and pQE-80L-Kan glf into E. coli. This is approved by previous research using BL21 (P450BM-3 QM/GlcDH/GLF) for α-pinene biotransformation (2). The following step to lyse α-pinene oxide used E. coli with pET-28a prα-pol introduced, which is also approved by previous study (3). Since the target gene used in our project has been described in detail in previous research, it is proved that our project is safe to a certain extend.

We carefully considered the choice of organic solvent. Previous research dissolved α-pinene and α-pinene oxide with hexadecane, which may be fatal if swallowed or enters airways [Danger Aspiration Hazard] (4). Therefore, we used corn oil instead, which has been proved to be a relative excellent organic solvent (5). It has been verified by the U.S. Environmental Protection Agency (EPA) to be safe for both human health and the environment (6).

For safety reasons, we also did preliminary experiments. We tested the solubility of α-pinene in corn oil. The results showed that α-pinene had high solubility in corn oil. We also conducted a series of experiments to investigate the sensitivity of our engineering bacteria to α-pinene or α-pinene oxide. See the Results section for details.

Product Structure

With awareness of biosafety in mind, we considered monitoring techniques, control of the release and waste management in the design of product structure.

The key part of our product is a kind of biological filter with our engineering bacteria fixed in. This film will be covered by a certain material which can isolate bacteria from the outside air, preventing bacteria from spreading into the environment. Additionally, we intend to use a biosensor that can monitor the accidental release of E. coli rapidly (7). When the concentration of E. coli in the air is higher than the threshold, it will alarm, indicating the leak of microbial agents. After usage, the unit section including membrane and covering material will be returned back to factory and autoclaved as scheduled.

Figure 2. Summary of product structure regarding biosafety

Biosafety Handbook

Based on our project, we wrote a Biosafety Handbook. The aim is to popularize biosafety to the public, while emphasizing the security of our products. This handbook first gives a basic introduction to bacteria especially engineering bacteria. Then it explains the potential harm of bacterial agents to human and environment. Therefore, we integrated how we operate safely in laboratory and how we design our products to achieve biosafety. We are trying to raise public biosafety literacy with this handbook. The iZJU-China team hopes that we can live in harmony with microorganisms and create a better home together.