Overview

Biosynthesis plays an important role in today's aspirations to produce materials that people need without relying on petroleum fuels. In this context, our project aims to develop an innovative biosynthetic pathway to produce lycopene and patchoulol and to use our products as dyes. Our project involves the use of microorganisms modified by a genomic introduction of heterologous genes, so controlling the resulting GMOs is crucial because we cannot determine the impact if they were released from the laboratory. In addition, we have taken into account the security issues that may exist in the course of carrying out the project work, and set up preventive measures for these problems. We will explain in this part how our project has been thought to be respectful of the legislation and safe.

Laws and Regulations

We have considered the different legislation regarding the biosafety for the use of biotechnology and its products in China. At the same time, we didn't ignore the laboratory safety rule. After analyzed the Chinese laws and Laboratory Safety Manual, we complied with them strictly during the conduct of our project.

Safe Project Design

Safety to Humans

We have known that some pathogenic bacteria may pose a health risk to the experimenter during the experiment, but our project is based on a non-pathogenic strain of Pichia Yeast with low survivability outside laboratory conditions. We have chosen this microorganism for their superiority in heterologous expression, but also for the low risk they present for human safety and the environment.

According to American Type Culture Collection(ATCC), microorganisms are classified into four categories based on their health risks to humans and the environment, each of which corresponds to four levels of laboratory treatment.

Pichia pastoris are classified in the security Level 1 by the ATCC and therefore the harm to operator and environment is almost negligible.

After excluding the toxicity of the engineered bacteria itself, we thought about whether it is possible to produce toxic molecules in the production process. Therefore, we have checked the toxicity of each molecule. For each molecule with a potential hazard, we have searched for the quantity presenting a danger in the literature. In our products, lycopene is widely used in the food industry. It is not only non-toxic to human body, but also beneficial to human health. And in the acute toxicity study on mice, the LD50 of patchouli alcohol was 4.693 g/kg^[1], which was evaluated as low toxicity and safe. As for the intermediate products in the production process, because the yield is very low, and most of them are harmless to human body, so it is impossible to cause harm to human body.

We have also estimated the maximal quantity of molecules we could produce in a 1 L culture. Even if one person takes in this whole quantity, the amounts are way under the toxicity threshold.

Therefore, we conclude that the products are produced in quantities far below the concentrations at which they are toxic. Therefore, the production of these molecules is harmless to the experimenter.

Safety to the Environment

We have considered the possibility of horizontal transfer of genes after the escape of the modified strains from the laboratory, but for various reasons, we didn't set a kill switch for our strains. The primary reason is that the addition of the kill switch will increase the burden of the modified strain, which may adversely affect our production. In addition, our strains will be used in factories, where they will function in fermentation tanks. Thus, the possibility of the strains escaping is low and the modified gene in our strain will be less likely to contaminate the natural strain.

If the project goes ahead, our products will be released outside the laboratory as dyes after being approved, but not include our modified strains. It further ensures that our engineered strains do not lead to uncontrollable risks.

In the dyeing process, due to the use of toxic chemicals such as acid, alkali, bleach and heavy metal ions, serious water pollution will be generated during the traditional dyeing process. Although we phase in the laboratory using ethyl acetate as solvent, but we envision when put into industrial production, the use of a no harm to the environment or less harmful solvents to dissolve the dye molecules, in this case, FUNCDEYS won't produce large amounts of toxic chemicals and water pollution can be avoided to some extent.

Safe Lab Work

Although we were only required to conduct our experiments in a Level 1 lab, each of our team members received thorough safety training before entering the lab. Only the people who have had previous training in lab equipment and safety protocols were allowed to perform experiments. We were also guided by at least one of the instructors during our experiments.

Safety to Humans

In order to ensure the safety of experimenters during the experiments, we all learned how to operate the laboratory instruments and the procedure of storing the experimental reagents. Safety issues that need to be paid attention to when using instruments were posted near the instruments in our laboratory to prevent instrument operation mistakes.

During the experimental, the organisms were be stored inside a freezer. For long term storage, the culture was frozen and left at -80°C. High-risk chemicals were kept in safety cabinets and only when they need to be used do we unlock the door to remove them and put them back immediately after use.

In addition, we also participated in fire drills to clarify what we should do to escape in the event of a fire accident.

Safety to the Environment

Considering that the modified bacteria may be transmitted through physical media (such as the experimenter's clothing, body, etc.) and that improper waste disposal may also cause leakage of the modified bacteria, we have strict rules for the regulated operation and waste disposal process during the experiment. Our members have also received comprehensive experimental training to reduce the possibility of leakage in the laboratory as much as possible.

During our experiments, we strictly follow the experimental code and wear gloves for all experimental operations and prevent other body parts from touching the experimental area. At the end of our experiments, we discard our gloves and experimental materials in a timely manner and autoclave the reused equipment at 121°C, 1 bar pressure. All of our experimental waste and domestic waste are placed and disposed of separately, and experimental waste was stored in a sealed bin, with special symbols displayed, until removal by a waste contractor for disposal.

Safe Human Practice

In Human Practice, we hope to get real voice of the society and improve our programs from the feedback of people from all walks of life. However, while ensuring that our feedback is effective, we should also respect and protect our interviewees' privacy and information security.

We have informed the interviewees of our general interview content and form before the interview, and only after receiving their consent did we continue our interview. All the people we collected comments and feedback have read our informed consent form and signed a consent agreement. After each interview, we asked the interviewee if they would allow us to post photos and conversations on the wiki, and sent the interview documents we had recorded to the interviewee. If they think there is something inappropriate or they don't want to be put on the wiki, we will rework it or remove it. Therefore, although some of the feedback and suggestions were not convenient to display on Human Practice, they have been incorporated into our project.

COVID-19

Unfortunately, COVID-19 pandemic was still running during the course of our project. Our team also conducted comprehensive vaccinations to reduce the risk of infection. On the first day in the laboratory, each student needs to submit a screenshot of his health green code and travel record. If he returns after leaving Guangzhou during the period, he needs to re-collect these two screenshots.

During our human practice, we also always remember to maintain safe personal protection. We try to choose a time and place for offline communication interviews where there are no recent positive cases, and if this is not possible, we conduct our human practice in an online manner. During offline communication, we also take care to always wear masks, maintain appropriate social distance and disinfect with alcohol in a timely manner.

Fortunately, we did not suffer from any contamination during the course of the competition.

Reference:
[1] He Jingjin, Peng Shaozhong, Xie Qingfeng, Li Yucui, Su JiYan, Lai XiaoPing & Su Ziren.(2012). Study on the Acute Toxicity of Patchoulol. Shi Zhen National medicine and national medicine (02),274-275.