Workspace
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E. coli DH5α
DH5α is optimal for stable amplification of the DNA materials. It is a non-mutagenic strain of E. coli and is the most commonly used K-12 laboratory strain. This is the bacterial strain that we used for storage and cloning. This strain is non-pathogenic and non-colonizing to animals and humans, contains disabling mutations and are highly unlikely to survive in the human guts and environment. DH5α also does not produce chemical toxins and does not cause any acute or delayed symptoms and effects. The strain also poses minimal potential hazard to laboratory personnel and the environment, not known to consistently cause disease in immunocompetent adult humans and only require the BSL-1 containment and standard microbiological practices for experiments using this strain.
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E. coli BL21
The E. coli BL21 strain is harmless and widely used in recombinant protein production due to it being an endotoxin-free strain of E. coli. It is also used for therapeutic proteins in medical applications because it is harmless and non-hazardous while having rapid growth and high expression rate. This strain has been engineered to only contain lipid IVA instead of full lipopolysaccharides which could increase the safety of the strain. LPS in high amounts can lead to fever, septic shock, and inflammation therefore the lack of it could make this strain to be utilized for OMV-based vaccines.
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E. coli BL21 (DE3)
One of the strains in B lineage of E. coli. One of the most widely used strains in high-level expression of recombinant proteins. Lacking the lon and OmpT membrane protease, it degrades less heterologous proteins that are expressed by the cells.
Before starting our work in the laboratory, we got a safety and risk workshop from the iGEM Ambassadors in June. Any laboratory activities related to the project were also overseen by our team’s primary and secondary PI from direct consultation and mentorship.
All of our activities in the lab follow the ISO 17025: 2017 guide for the standard laboratory procedure, and also supported by the ISO 9001: 2008 for the laboratory quality management. The ISO 9001: 2008 manages the arrangement of hazards, reducing risks, and maintaining a safe environment for researchers in the laboratory. There should be separated areas for protein/RNA/DNA working space and trash bins according to their categories to reduce the risk for contamination either to humans or the environment.
In the laboratory, a safer alternative to EtBr for nucleic acid visualization is used, that is the GelRed. EtBr is carcinogenic and a potent mutagen, and is able to be absorbed by the skin and cause irritation if inhaled. The use of GelRed also eliminates the need for UV lights, therefore we use GelRed for our experiments.
Standard laboratory procedures are also implemented including wearing lab coats inside of the lab to protect us from potentially harmful materials and reagents, taking off coats when going outside of the lab to prevent cross contamination, the use of latex gloves and rubber footwear to avoid contact with the chemicals and electricity, and also to avoid damaging the enzymes such as RNAse, DNAse, and other biological materials. Cultivating microorganisms on a clean bench space or the Laminar Air Flow (LAF) to ensure the equipment are sterile and less risks exposed to the researchers. After working with the cultures, they will go through the destruction process to destroy the microorganisms in the glassware and to inactivate chemicals before being thrown away as waste to a specific category of trash bin.
Strains Safety
It is important to ensure the safety of our strains before working on it in the laboratory. To prevent illness from the microorganisms and prevent cross-contamination to the environment, we chose non-pathogenic chassisses that do not contain any potential harmful risks to the researchers themselves, or even the environment. Working safely and securely are important key aspects for a good lab practice. Precautions have been analyzed during our preliminary safety requirements before getting into the lab that can be found in our safety form. The CHOP system uses the E. coli DH5a, BL21, BL21(DE3) strains and only needs BSL-1 infrastructure.
COVID-19 Regulations
Because of the COVID-19 pandemic, there were additional regulations we needed to comply with when working inside of the lab. The regulations include the reduced maximum capacity of people working in the lab to 50% (around 4-5 people for one period of time), submitting our COVID-19 antigen testing before getting into the lab, using a mask or double masks during our work in the lab, book materials and equipment needed the day before to avoid crowding, and only able to conduct experiments during our schedule and is only one person from the team is able to work. Due to the restriction that only one person can work inside of the lab from each team, we decided to use two different labs in our university for efficiency and to be able to work on as many experiments as we can with the time limit we have.