Our project is to efficiently express the glucose-lowering peptide Vglycin through biological synthesis in Pichia pastoris. The process of biological experiments inevitably involves safety issues, and it is crucial to ensure the safety of the operators and the environment. We conduct our safety work in the following aspects.

Biosafety

We choose Pichia pastoris as the host to produce peptide Vglycin, which is human- and environment- friendly organism.

Experimental safety

In the experiment, we strictly adhere to the laws of standard operating procedure (SOP) to avoid damage to the instruments and the safety of operators, and to prevent drug leakage.

Safety training

The lab instructor provides us with experimental safety training and offers suggestions when we carry out the experiments.

Biosecurity measures

Based on the biosecurity risk assessment of our project in the lab, we developed a series of biosecurity measures to ensure the safety, such as physical biosecurity, information security and cybersecurity, information security.

All the parts we used in this project were all synthesized by a bio company and cloned in the vector that listed the Part library of iGEM. The plasmid DNA was amplified in DH5a, Vglycin was induced expression in Pichia pastoris, which also existed the iGEM whitelist. DH5a and Pichia pastoris are classified in RISK GROUP 1. Therefore, there is virtually no risk to human health or the environment.

Pichia pastoris is a eukaryotic microbe that belongs to risk Group 1 in the iGEM whitelist and is not harmful to human health. We have verified the safety of Pichia pastoris through the appropriate authorities, who confirmed its use^[1]. The culture is slowly frozen and stored in a -80℃ refrigerator. We wear gloves and masks to protect ourselves during the transformation operation. The transformation vector we use is a plasmid, which is non-toxic and safe^[2]. During the experimental operations that require cryogenic preservation, items are processed at low temperatures. Once done, they are quickly returned to the refrigerator for storage.

Figure 1. Our laboratory pictures.

Our laboratories are clean and well-equipped with autoclaves, ovens, various freezers and biosafety cabinets (Figure 1). We wear lab coats when entering the lab and refrain from wearing slippers. Females tie their hair back to avoid it getting caught in equipment or coming into contact with hazardous materials.

We conduct experiments in strict accordance with laboratory safety guidance. For all the experimental operations, we followed the experiment.

⚫ During the experiment of our project, we will use some toxic reagents such as formaldehyde, Ethidium Bromide (EB), TEMED, PAM, etc. In response to these toxic reagents, we will take the following measures:

1.Formaldehyde:Wear appropriate personal protective equipment when using, such as gloves, protective glasses and masks. Ensure that the laboratory has a good ventilation system to reduce formaldehyde concentration. If formaldehyde leaks, clean it up with an appropriate absorbent and ensure the area is well ventilated.

2.Ethidium Bromide (EB) :Avoid direct contact with EB and wear disposable gloves and protective glasses when operating. After using EB, the solution containing EB should be purified and disposed of to prevent environmental pollution and human health hazards.

3.TEMED：Operate TEMED in a well-ventilated laboratory. Avoid direct contact with skin and eyes. Wear protective clothing, chemical safety glasses, and rubber gloves during operation. Handle the TEMED with caution to prevent aspiration, perform the TEMED quickly, and store it in a sealed container.

4.Polyacrylamide (PAM):PAM itself is generally considered non-toxic, but inhalation of dust should be avoided during operation. Wear gloves and goggles when operating. Waste should be properly disposed of after use to avoid direct discharge into water sources.

⚫ For the instruments used in the process of our project, such as centrifuges, sterilizing pots, fume hoods, etc., there will also be some security risks during their use. In response, we have listed their security practices.

1.Centrifuges: We have standard operating procedure (SOP).

2.Autoclaves:We have standard operating procedure (SOP).

3.Ovens:We have standard operating procedure (SOP).

4.Clean benches:We have standard operating procedure (SOP).

5.Fume hoods:We have standard operating procedure (SOP).

6.Ultraviolet analyzers:We have standard operating procedure (SOP).

7.Biosafety cabinet: We have standard operating procedure (SOP).

Before our experiment, we conducted laboratory safety training, including experimental operation and experimental instruments, and team members were required to pass certain exams before entering the laboratory for experiments. In addition, at the beginning of the experiment, we were divided into groups of two or three, so that we could supervise each other during the experiment and prevent misoperation.

Before entering the lab, our instructor provided us with general knowledge of lab safety and how to use the instruments.

Regarding the Pichia pastoris we use, our treatment includes:

⚫ Aseptic operation: The SOP must be strictly followed during the entire experimental process. Operations are conducted in the clean bench, and the use of appliances must be sterilized to prevent contamination by stray bacteria that could affect the results of the experiment.

⚫ Autoclaves:Used to sterilize media, yeast cells, and other biological materials used in the experiments to ensure that all bioactive substances are completely destroyed.

⚫ Temperature control:Pichia pastoris usually grows well at 28-30oC, and the temperature should be stable and uniform during the cultivation process.

⚫ Chemical disinfection:For waste that cannot be treated by high-temperature sterilization, we use chemical disinfectants. For example, use a 75% ethanol solution to wipe and disinfect the experimental table and equipment.

A biosecurity risk assessment was carried out based on our project proposal before we started. According to this assessment, we developed a series of risk control measures. In this section, measures are presented for different biosecurity areas to enable facilities to implement locally effective and sustainable biosecurity risk control measures to manage biosecurity risks.

Physical biosecurity

Physical biosecurity is intended to prevent the misuse, loss, or theft of biological agents^[3].All the samples and reagents stocked in the freezers were double locked and under security of two people. Anyone who works alone was not permitted. We also have the regulations of physical access to lab with relevant information and restriction to authorized personnel.

Information security and cybersecurity

Similar to physical security, data protection and cybersecurity include regulated physical access to information, for example, digital and physical access to computers and devices, including all the laboratory equipment that we used for our project were not permitted to access with personal account and password. For the device with network capability, password protection of information and regulation on data on portable devices. Information with biosecurity relevance is identified by the biosecurity risk assessment and requires biosecurity risk control measures. Those measures are outlined in the following subsections^[4-5].