1.Safety Assessment
We have carefully researched and studied the safety protocols and have categorized our lab as BSL-1. During the lab process, we strictly followed the safety rules in our university lab.
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All the members of our team attended the lab safety course and took the lab safety test, and only those who passed the lab safety exams were allowed to conduct the experiments.
Safety warning signs can be seen everywhere in the lab, reminding us to pay attention to safety at any time of the day.
2.Operation Safety
2.1 Training
Before entering the lab, we received obligatory procedural training to ensure the safe environment in it, which included basic protocols and proper handling procedures for ourselves and others. Subsequently, we completed specific training for each instrumentation before operation. Additionally, we were required to review the operating instructions and get further instructions from our instructor before utilizing any equipment. When operating the equipment, we ensured that we strictly follow the instructions, remain continuously present when it was in use, and promptly turn it off once we finished. Also, conducting cellular experiments alone mandated performing a special test.
2.2 Wearing
Team members were required to wear proper protective gear, such as lab coats, masks, goggles, and gloves, to ensure their safety prior to entering the lab. Furthermore, shorts and slippers are strictly prohibited inside the lab.
The requirements for the cellular lab are even stricter. When going into it, individuals must wear standardized masks and gloves, and further get changed into lab coats and sterilized slippers designated for this lab. They should tuck their sleeves into their gloves and secure them with rubber bands to minimize exposing their skin. All staff members must follow these procedures to keep the environment secure and in control. Individuals with open wounds should not enter the cell room in order to protect themselves as well as the cells.
2.3 Sterility
Our experiments are performed primarily in biological safety cabinets to preserve sterility and minimize contamination. Before conducting the experiment, the UV light is activated for at least thirty minutes for sterilization. Prior to the experiment, we clean the countertop with 75% ethanol multiple times. Additionally, hands and all the equipment should be sprayed with 75% ethanol before we enter the bio-safety cabinet. While conducting the experiment, an alcohol burner was ignited inside the bio-safety cabinet and all our operations were performed within 10 cm of its flame. When the experiment is completed, the countertop is sanitized and wiped multiple times using 75% ethanol and the UV lamp is switched on for a minimum of thirty minutes.
More specifically, in the cell chamber, we have established a buffer area for UV sterilization with no presence of any personnel. Experimental materials must be irradiated with UV for a minimum of 30 minutes before being brought into the cell chamber to guarantee sterility.
3. Material Safety
3.1 General Reagents
All general reagents are categorized and stored in specific reagent cabinets in a cool and well-ventilated environment to avoid direct exposure to sunlight. The storage area should be organized in a reasonable manner and receive regular checks and registrations.
3.2 Hazardous Reagents
Flammable, explosive, and toxic chemicals, as well as other hazardous substances, are stored in designated reagent cabinets as mandated by relevant regulations. Labels and warning signs are posted for identification. To ensure safety, a “two-person, two-lock system” principle is implemented for obtaining these materials and any removal must be promptly recorded. At the same time, laboratory staff are knowledgeable of the hazardous properties of the chemicals used, safety and protection procedures, methods for storage and waste disposal, as well as emergency response protocols.
4. Waste Disposal
4.1 Liquid waste
We collect waste liquids by category. Waste liquids are collected in 25L waste liquid tanks, which need to be labeled with instructions. After dumping the waste liquid, the container should be closed immediately and get registered. The tanks can only be filled up to 80%, and when its containing liquid reaches the standard, an appointment should be made so that professional staff can come and collect it.
For bio-waste, such as polluted liquid medium, rather than simply pouring it into the drain, we use an autoclave to sterilize it before transferring it to a waste tank for regular disposal by a professional company. This method reduces the risk that microorganisms and genes leak into the environment.
Outside the lab, there is an outdoor sewage disposal system, which can initially treat the waste liquid discharged from the lab, avoiding its direct access into nature. At intervals, particular staffs will conduct sampling to test the water quality.
4.2 Solid waste
Solid waste generated in the experiment, such as used pipette tips, 96-well plates and cell culture flasks, should be dumped into a special garbage can which filled with yellow plastic bags. Sharp objects, such as needles and syringes, should end up in disposable sharps boxes. Waste also needs to be dealt with by staff for on-site collection.
4.3 Empty bottles
We have specific boxes for collecting empty bottles and broken glass to ensure safety and prevent the leakage the chemicals. Hazardous waste labels are posted outside the cartons where empty bottles are collected. When the box is full, the professional staff will come and collect them together.
4.4 Waste declaration method
This is the official reservation mini program for waste declaration.
5. Emergency Facilities
The laboratory is outfitted with appropriate fire suppression equipment, emergency showers, eyewash stations, and first aid kits that are regularly serviced.
6.Microorganisms safety
The microorganisms used in our experiment met the iGEM competition’s official whitelist requirements. To stick to safety more rigorously, we consulted with the competition’s HQ via email and received a positive confirmation. We mainly used the following microorganisms:
6.1 Bifidobacterium longum
Bifidobacterium longum (BL) is a probiotic that exists in the human intestinal tract and is not harmful to human body. In addition, it is a strictly anaerobic bacterium that cannot survive in an aerobic environment and does no harm to the environment. Therefore, Bifidobacterium longum will pose no threat to the personnel or the environment during our operations in the lab. We use a BL type strain, ATCC15707, in the experiments.
6.2 Escherichia coli
For the convenience of operation and higher success rate, we used E. coli DH5α and BL21 for plasmid amplification and expression respectively during the experiment. Both of them are relatively safe and are only used in preliminary experiments and will not be put into the final product. Due to the harsh culture conditions and low electroporation of BL, most of our interest proteins were produced by E. coli at first.
6.3 Fusobacterium nucleatum
Fusobacterium nucleatum is a pathogenic bacterium, which is an important factor in inducing colorectal carcinogenesis and inflammatory cancer transformation. So, it is inevitably needed in the experiment. In the consideration of safety, we chose the strain ATCC10953, which is less pathogenic and conforms to the bio-safety level 1 standard.
certification: CLICK HERE
6.4 Colorectal cancer cell
We used colorectal cancer cells SW480 to verify the anticancer efficiency of the antimicrobial peptide, which was also only used in the experiment. The SW480 is proven to be pathogen-free and HQ said “it can be used without the need to submit a check-in form”.
7. Dry lab safety
Our dry lab work was mainly conducted on these two studios. Passwords are needed if the operator wants to enter them. In addition, to prevent hackers from stealing experiment data, studios are forbidden to connect to the Internet. To avoid the studios from being invaded by viruses, only specific CDs can be used to copy data from studios.
8. Risk Management
8.1 Equipment usage
Each piece of equipment in the lab is managed by a specially-assigned person and every piece has a detailed Standard Operating Procedures (SOPs). All members must obey the SOPs to reduce potential safety hazards during our use. If you want to read our equipment SOPs, please click
HERE
8.2 Safety guidance
Not in our team roster as he is, Peiwei Yang has helped us manage a lot of safety issues. He is the general person in charge of lab safety. He gave us safety training before and during the experiment, taught us how to use each instrument and informed us of the detailed that need special attention.
8.3 Inventory controls
One of our instructors, Weiyan Qi, is in charge of the lab inventory controls. She keeps the key of inventory. Once we need any lab consumable, we should tell her and fill the record form. Good Inventory controls can prevent material shortages and help us conduct experiments more effectively.
