General Lab Safety Rules

We observed and followed all the general laboratory safety rules under the guidance of Professor Woorin Lee, as enumerated below:

1. The location of fire extinguishers, safety showers, and emergency exits was documented.
2. Food and beverages were never consumed in the laboratory.
3. We donned protective gloves, closed-toed shoes, and lab coats to prevent chemical substances from contaminating the skin and eyes.
4. To reduce chemical exposure, students tied their hair back.
5. All of the students behave maturely and responsibly, without any horseplay.
6. We complied with the disclosure standards outlined in "Standards for Disposal of Household Waste and Designated Waste."
7. We only exited the laboratory when all experiments had been completed and no hazardous procedures were ongoing.
8. We never recycled chemicals or containers.
9. We were instructed to report minor accidents and errors.
10. Before we utilized the equipment, the instructor provided detailed instructions.
11. We managed physical access to the lab and storage spaces.
12. We regulated data entry into devices and databases.
13. Before waste departed our laboratory, we oversaw waste management systems such as decontamination.
14. We were instructed thoroughly by the professor before starting any lab experiments.
15. We were informed about emergency procedures.
16. We learned about disinfection and sterilization procedures.
17. We were informed about chemical, fire, and electrical safety during training.

Project Safety

This iGEM project aims to genetically modify HEK293 cells to synthesize afamin and Wnt3a proteins within the cell culture medium. We hypothesize that this modified medium could serve as a potential substitute for fetal bovine serum (FBS) in the production of lab-grown meat, thus maximizing procedural efficacy while mitigating costs and ethical concerns. By engineering HEK293 cells to produce afamin and Wnt3a, we anticipate achieving a more cost-effective and sustainable approach to cultivating lab-grown meat. Please refer to the provisions below to review our team’s compliance with protocols issued by iGEM’s Safety & Security Committee and the World Health Organization (WHO) standard guideline.

Specific Materials Posing Potential Risks

• Escherichia coli (DH5-alpha) was utilized to amplify the vectors containing afamin and Wnt3a genes.
• Afamin and Wnt3a gene-containing vectors were transfected into HEK293 cells.
• The conditioned media (enriched in Afamine and Wnt3a proteins) produced from the HEK293 cells were collected and treated on the bovine muscle cell line (MyoB).
• Lipofectamine transfection reagents were used for the transfection process.
• Afamin and Wnt3a proteins were overexpressed in the HEK293 cells.
• Sodium dodecyl sulfate (SDS) and polyacrylamide were used for the western blot analysis of afamin and Wnt3a proteins.
• Afamin and Wnt3a were enriched and used in our conditioned media to the bovine muscle cell line (MyoB) to test cell proliferation.

Specific Activities Posing Potential Risks

• Pipetting and Liquid Handling: Accidental spills, splashes, or aerosol generation can occur when pipetting or transferring infectious materials, potentially leading to exposure.
• Centrifugation: Centrifuging infectious samples can create aerosols if not properly sealed and balanced, increasing the risk of inhalation or contact exposure.
• Laboratory Equipment Use: Improper use or maintenance of equipment such as incubators, biosafety cabinets, and autoclaves can lead to equipment failures or contamination incidents.
• Working with Sharp Instruments: Handling sharp instruments like needles and scalpels poses a risk of accidental punctures or cuts, potentially leading to infection.
• Spill and Leak Response: Improper management of spills or leaks during experiments can lead to contamination of the laboratory environment and pose risks to laboratory personnel.
• Storage and Transport: Inadequate storage and transportation of infectious materials can result in accidental exposure if containers break or leak.

Specific Implementations Posing Potential Risks

If this concept becomes a completed product, it will be utilized on a large scale. This necessitates a safety and security risk assessment that addresses the risk posed by a broader application. Due to the absence of synthesized DNA/RNA, we do not anticipate any autonomous dissemination into the environment.

Risk Management Provisions

Tissue culture detritus was deactivated in a 10,000 ppm hypochlorite solution for at least two hours before disposal at the sink with excess water. In addition, contaminated pipettes were saturated overnight in a 2,500 ppm hypochlorite solution before being incinerated. In general, the following recommendations were used to resolve cell culture waste.

1. The contaminated culture should be autoclaved or disinfected and then discarded.
2. Clean and disinfect all incubators, centrifuges, refrigerators, microscope stages, and other equipment, including pipettors, that came into contact with cell culture detritus.
3. All media, media components, and other reagents used with the contaminated cell lines must be discarded, and all other cell lines in the laboratory must be quarantined and tested for mycoplasma to detect the spread of contamination.
4. To ensure the safety of the experiment, all members utilized biosafety apparatus, received project-specific safety and security training, and developed a comprehensive communication plan.
5. When entering the laboratory, all members and the instructor were given personal protection equipment, such as lab coats and gloves.
6. Prior to conducting experiments, participants were educated on emergency procedures, lab access, and disinfection to ensure their safety and that of the supervisor.

Compliance with iGEM Security Procedures

1. "Do Not Release" Policy: Our wet lab initiatives, including sources or products of genetically engineered organisms, were not utilized outside the lab and restricted to the experimental phase.
2. "No Human Experimentation" Policy: Our initiative does not involve human testing or direct human contact, and no human samples (swabs, blood, etc.) are required.
3. "Whitelist" Policy: Our project does not include animals, parts, or materials outside the scope of the Whitelist (or those from higher-order vertebrates), as all parts and organisms are sourced from reputable commercial and academic suppliers.
4. Policy Regarding "Human Subjects Research": Our team does not conduct surveys, interviews, or other human subjects research within the context of wet lab experiments.
5. Our workspace consists of an exposed bench and biosafety cabinet.
6. We have cell culture room space and laboratory lecture space since the biosafety level of our workspace is Level 2 (moderate containment) according to iGEM’s risk group classification.
7. The WHO Manual on Biosafety in Laboratories: Our initiative implemented biological safety fundamentals and safely handled biological agents in laboratories.

Anticipating Future Risk

1. Future expansion of our undertaking would not necessitate release beyond containment.
2. Because our engineered conditioned medium is incapable of spreading to the environment, our conditioned media poses no future threat to agricultural animals, crops, or domesticated animals (e.g., from pathogens or ecological disturbances).
3. Our project is fundamental; we have no specific real-world application in view (e.g., a library of standardized promoters or a system for cell communication).