Molecular Biology Lab
All lab members have undergone and passed safety training given by the lab technicians before doing any lab work. This includes safety policies and rules of the labs. For example, familiarize themselves with the location and proper use of fire extinguishers, fire blankets, and other equipment in fire safety boxes. They have also learned how to operate emergency showers and eyewash stations correctly. They learned how to open and close the doors of cold rooms and warm rooms correctly, and are aware of the location and application scenarios for emergency call buttons.
While in the laboratory, members always wear lab coats, long pants, and shoes that cover their toes. Members with long hair will tie their hair back.
Most organisms and DNA fragments the team used and generated are in the iGEM 2024 White List. For fragments that are excluded from the White List (CRISPR/RNAi, nsp1234(VEE), and nsp10/14 (COVID)), a check-in form was submitted for approval.
When handling vessels that have just been removed from high-temperature devices such as autoclave rooms, microwave ovens, and water baths, members wear heat-resistant gloves to protect against heat and to prevent the vessel from breaking due to improper handling.
Fire safety is guaranteed when operating around alcohol lamps, and all team members have mastered the skills to handle spillage.
During agarose gel electrophoresis experiments, members wear gloves when handling DNA dye and gels stained with the dye, as the dye (such as Sybr Safe) is carcinogenic. They also ensure that containers that have come into contact with the gel are cleaned thoroughly after use. Waste gels from visualization are processed separately from general waste.
For handling high-concentration gel (over 2%), run it in a cold room to prevent fires due to heat generated from high currents.
When working with liquid nitrogen and -80°C freezers, it is required to have two people present to assist with the operation. Wear heat-resistant gloves to prevent frostbite when in direct contact.
Cell Lab
Members responsible for cell culture have received training on the safe operation of biosafety cabinets (cell hoods) given by the Ph.D. student in the lab. This includes:
- 1. Spray items with alcohol before bringing them into the hood. Spray alcohol on hands and forearms for disinfection
- 2. Turn on the UV light before and after use
- 3. Wearing lab coats that fully cover the skin
- 4. At the end of the experiment, add bleach to the designated beaker in the hood, allowing it to react fully with the waste liquid before being absorbed into the waste tank.
Tips, tissues, and Eppendorf tubes that have come into contact with cells or E. coli are disposed of in biohazard waste (separate from general waste), for further processing. Sharp debris is placed in a special container.
DNA Fragment Safety
CRISPR, leveraging its gene drive capabilities, can significantly alter the genetic inheritance patterns in organisms, thereby posing ecological risks. RNAi, while useful for gene suppression, may also lead to off-target effects. To ensure biosafety, the team used TMP (tetracycline derivative) as an inducer to control the activity of the CRISPR system. The destabilizing domain (DD) was fused to the Cas9 protein to construct an inducible CRISPR/Cas9 system. In the absence of TMP, the DD-Cas9 protein will be degraded by the proteasome, while in the presence of TMP, the DD-Cas9 protein is stable, thereby achieving control of CRISPR/Cas9 activity.
DNA sequences such as nsp1,2,3,4 from Venezuelan Equine Encephalitis (VEE) and nsp10/14 from SARS-CoV-2, the virus responsible for COVID-19, are not included on the White List due to the biosecurity risks associated with these pathogens. The team only used the self-replicating and proofreading domains to obtain their function as a genetic tool while ensuring biosafety. All experimental materials and waste are handled and disposed of under biohazard guidelines.
All experimental materials and waste are handled and disposed of under biohazard guidelines.
Microfluidics Labs
- Proper training was given to members before the start of work with the laboratory equipment.
- While doing experiments in this lab, lab coats were always worn by members. Long hair was required to be tied back.
- Safety gloves were worn at all times during the experiments using the protein nanoparticles.
- Members working in this lab have been properly trained to use the lab equipment.
- While doing experiments in this lab, members always wear lab coats. Members with long hair are required to tie their hair
- Proper training was given to members before the start of work with the laboratory equipment.
- While doing experiments in this lab, lab coats were always worn by members.
- Long hair was required to be tied back.
- Safety gloves were worn at all times in preparation of the nanostructured lipid carriers.
- During the usage of the Quant-iT RiboGreen RNA Assay Kit for experimentation, it was handled with care as it may be considered a potential mutagen.
- The solution containing the Ribogreen reagent was also properly disposed of.
References
Rona, G., Zeke, A., Miwatani-Minter, B. et al. The NSP14/NSP10 RNA repair complex as a Pan-coronavirus therapeutic target. Cell Death Differ 29, 285–292 (2022). https://doi.org/10.1038/s41418-021-00900-1
Zimmermann, L., Zhao, X., Makroczyova, J. et al. SARS-CoV-2 nsp3 and nsp4 are minimal constituents of a pore-spanning replication organelle. Nat Commun 14, 7894 (2023). https://doi.org/10.1038/s41467-023-43666-5