Safety
Nip unsafe factors in the bud -- The Book of Changes
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When it comes to genetically modified organisms, safety is the primary and acute issue. Therefore, strict experimental procedures and standardized experimental spaces are crucial for ensuring personal, social, and natural safety. We have solved this problem from three aspects: project design, laboratory work, and shipping. The 2023 OUC-China team implemented the following security procedures. The safety form we have completed has been submitted and approved.
Project
Design
1. This year, our project used two types of organisms: XM01 Aspergillus niger and Escherichia coli BL21.
● Degradation of benzopyrene: Engineering yeast expresses Upo monooxygenase, combined with rhe red light system
● Synthesis of MEL: Optimization using gene knockout
● synthesis of α- Olefins, 10-hydroxydecanoic acid: Escherichia coli BL21
2. We use a specific reagent: kitchen waste oil
Our project involves Escherichia coli BL21 , M. aphidis XM01, and DH5 α. According to the policies of the China Center for Disease Control and Prevention and the iGEM definition, these two strains are classified as low potential risk group 1 (risk group 1 organisms do not cause disease in healthy adults). Among them, XM01 is not on the white list, and we have already registered it. In our wet laboratory, all gene transformation, vector expression, protein determination, and other operations are carried out in a biosafety cabinet and strictly comply with regulations. In order to protect the environment, all experimental waste was strictly sterilized.
Meanwhile, the kitchen waste oil used in our project is borrowed from nearby restaurants. It contains harmful substances such as benzene to propylene ratio. We have carefully studied the precautions for benzopyrene in MSDS, effectively avoiding the potential hazards of kitchen waste oil and preventing its pollution of the environment.
Figure 1 Laboratory Environment
Suicide
switch
In order to avoid the leakage of E. coli introducing foreign genes into the environment, we designed the NeuAc ribose switch. The sequence of NeuAc riboswitch was obtained through in vitro screening as reported by Schyung Cho. It consists of an RNA aptamer and a hammerhead ribozyme, as shown in the figure. The principle of its action is that NeuAc binds to the RNA aptamer, causing a conformational change in the entire switch, resulting in self cleavage of hammerhead ribozymes, creating a gap. Then, under the action of ReJ ribozyme in Escherichia coli cells, subsequent gene splicing is carried out, thereby reducing the abundance of downstream expressed genes' mRNA. To ensure the effective functioning of the switch, separate compartments have been added at both ends of the switch, with 5AAAAATAAAAAGAAAAA-3 at the 5 'end and 5-AAACAAAAAA-3 at the 3' end.
Through experiments, it has been shown that suicide switches effectively kill engineering bacteria and do not have an impact on the environment when leaked, so the design is reasonable. Meanwhile, all chassis organisms are disinfected before disposal to prevent gene leakage.
Figure 2 Design of Suicide Switch
Laboratory
work
We work in a clean laboratory called "Lab for Microinnovation and Enterprise", which is a BSL-1 laboratory. We carefully study and strictly follow the general guidelines for microbiology and biomedical laboratory biosafety established by the health industry standards of the People's Republic of China. (hyperlink: standard)
Before working in the laboratory, we receive experimental training. After iGEM pointed out that we should use biosafety cabinets instead of ultra clean workstations, we learned the difference between the two and understood that biosafety cabinets can protect the human body from pollution damage. Therefore, our genetic operations will be carried out in biosafety cabinets. (hyperlink: difference)
At the same time, we learned that there are specific instructions on the operation of hazardous chemicals in MSDS, so we learned about the section about benzopyrene, understood how to prevent harm to human body caused by benzopyrene, and how to self rescue in case of danger. We have effectively avoided the potential hazards of kitchen waste oil and prevented it from polluting the environment. (hyperlink: msds)
Figure 3 Laboratory Name and Laboratory Safety Regulations
In
cluding
● Brainstorm and spot problems
1. Chemical Safety - Benzo [a] pyrene
Figure 4 Benzo [a] pyrene hazard sign
According to the MSDS data sheet, benzo [a] pyrene may be fatal and can cause skin allergies, eye irritation, drowsiness or dizziness, genetic defects, and cancer if swallowed. It also poses great harm to the environment, especially toxic to aquatic organisms. Therefore, when operating with benzo [a] pyrene, attention must be paid to:
● Keep away from heat/sparks/open flames.
● Do not breathe dust/fume/gas/mist/vapor/spray.
● Do not allow contaminated work clothes to leave the workplace.
● Avoid release into the environment and clean up contaminated areas. Avoid generating dust.
● Wear nitrile rubber gloves/protective clothing/eye protection/face mask/respiratory protective equipment.
● Lock during storage.
● Suitable extinguishing agents: water, foam, carbon dioxide dry powder, without restrictions collect waste and spills. Strict waste disposal and container recycling, do not enter the sewer.
● Accidental inhalation: Immediately go to a place with fresh air for breathing and seek medical attention.
● Skin contact: Immediately remove all contaminated clothing. Rinse skin with water/shower. And consult a doctor.
● Eye contact: Rinse thoroughly with plenty of water. Call an ophthalmologist.
● Swallowing: Immediately call the detoxification center and have the patient drink up to two cups of water. Consult a doctor.
2. Microbial equipment safety
After official prompts and our own training, we learned the difference between biosafety cabinets and ultra clean workbenches. Biosafety cabinets are necessary sealing devices for handling potentially infectious materials, using high-efficiency particulate air (HEPA) filters to handle intake and exhaust. It can prevent exposure to particles and aerosols from biological agents. Provide personnel and environmental protection. Be careful not to use with volatile or flammable chemicals. We understand that ultra clean workbenches can only provide a sterile environment, and only biosafety cabinets can protect the human body from pollution and harm. Therefore, all operations involving microorganisms are carried out in biosafety cabinets.
3. microbial safety
We have checked the data from China CDC, NIH Guidelines, and American Type Culture Collection (ATCC) product sheets in detail to ensure that the strains we use are classified as low potential risk group 1, meaning they do not cause illness in healthy adults. The biological harm level of XM01 Mohs' black powder fungus is normal. Under normal circumstances, it propagates through multilateral budding, and in extreme cases, spores are produced. However, the experiment provides an environment with sufficient nutrition, so spores will not be produced and spread into the environment.
The laboratory also has corresponding facilities and safety equipment - biological safety cabinets; Personal protective equipment - protective clothing, gloves, safety glasses, respiratory protective devices, emergency flushing devices, etc.
All of our operations comply with the requirements of a first-class biosafety protection laboratory. All gene transformation, vector expression, protein determination, and other operations are carried out in a biosafety cabinet.
4. Data security
After official guidance, we have realized that data biosecurity risks are also an important issue, so we have carefully studied the relevant content.
The advancement of synthetic biology technology has significantly reduced the costs of gene sequencing, gene editing, and gene synthesis. The combination of synthetic biology and computer technology allows bioterrorism to create biological weapons and trigger real biological threats. Data biosecurity is a new field that combines biosecurity and cybersecurity, aimed at addressing or preventing unauthorized access to information and data in the life sciences and digital fields, as well as potential or actual malicious destruction, theft, and abuse.
Cyber biosecurity is a key pillar in protecting the bioeconomy, helping to improve national security, economic competitiveness, and social stability. Therefore, we should strengthen the importance of cybersecurity and prevent the misuse of key information, materials, and systems in the fusion of bioscience and cyberspace in a timely manner, in order to reduce the threat of biological weapon proliferation. Pay attention to data privacy, test data, and the integrity of public biological databases, automate the security monitoring of laboratory systems, timely assess relevant risks, and take early warning measures to ensure the maximum reduction of vulnerabilities in the life science industry, public health, and national security.
5. Basic Laboratory Rules
Behavioral safety:
● Equipped with experimental clothing, goggles, and disposable gloves, it is not allowed to wear experimental clothing outside the laboratory or in public places
● Tie long hair behind your head and do not wear short skirts, shorts, or open toed shoes
● Understand the location of safety equipment (eye wash, shower, and fire extinguisher)
● Always adhere to laboratory safety regulations, read instrument instructions, and read MSDS
● Write a sufficient experimental plan before starting the experiment
Do not eat or drink in the laboratory where chemicals are stored
Equipment safety:
● Properly operate emergency showers and eye washers, as well as other emergency equipment, including fire blankets, fire extinguishers, and emergency buttons
● Check if the equipment is working properly and do not use outdated or unsafe equipment
Chemical safety:
● Classify different chemicals and store them in appropriate locations through appropriate storage methods
● Use a fume hood to avoid the leakage of volatile toxic gases
Building safety:
● Remember the location of fire escape routes, plan fire evacuation, and obtain contact information for first aid personne
● Don't stay alone in the laboratory
Waste treatment:
● Deactivate all experimental microorganisms
● Classify garbage according to garbage disposal rules and dispose of it in an appropriate manner.
High pressure sterilization of biological waste before disposal; Chemical waste should be collected and handed over to professional personnel for treatment, and general waste should follow general treatment methods.
Figure 4 Reagent Storage as Required
Tra
ining
1. Training
All of us have received basic laboratory safety training for our university experimental courses. In addition, our mentor will spend a week ensuring that we acquire sufficient theoretical knowledge. The team leader who participated in iGEM in 2022 taught us a series of experiments to ensure that we gained sufficient practical experience.There are three main guidelines:
● Hardware: There should be necessary and functional experimental equipment.
● Personnel: All personnel conducting experiments must undergo strict training.
● Software: A sound regulatory system should be established.
The three policies complement each other and also help us achieve two basic goals. Ensure that all individuals conducting experiments are not infected and that the environment is not contaminated.
2. Law enforcement
Team members who violate safety rules will receive supervision and reminders from laboratory personnel until they are confident that they can independently complete the experiment. In the event of multiple violations or disregard for safety rules, the member will be required to write a critical self review, undergo retraining, and be prohibited from conducting any experiments for a month.
3.For the safety of the laboratory, each person conducts two routine inspections every day, which not only ensures the normal operation of our laboratory, such as providing experimental consumables and checking instrument operation, but also keeps away from non biological hazards, especially water and electricity. For the sake of efficiency, responsibility is assigned to each team member.
(1)Routine inspections must be conducted between 6:30-8:00 pm and 9:30-11:00 pm
(2)Laboratory cleaning must be done twice a week, and the floor must be wiped with disinfectant at least 3 times.
Figure 5 Our team members follow safty rules when doing experiments
Safety
form
We fill out this form within the specified time, submit it to the official website, and obtain approval (add attachment)