Strict adherence to laboratory instructions and safety protocols is paramount in bioengineering and synthetic biology. Our team prioritizes safety in every experiment, ensuring compliance with biosafety regulations and overall project safety guidelines.
Before commencing any lab work, all team members undergo comprehensive safety training. This training emphasizes the importance of adhering to safety rules, including the prohibition of eating and drinking in the lab and the requirement never to work alone. During all experimental procedures, team members are also instructed to wear appropriate personal protective equipment (PPE), such as lab coats, gloves, and goggles.
Our experiments are conducted in a Biosafety Level 1 (BSL-1) laboratory. Operations involving bacteria and volatile chemicals are performed within biosafety cabinets and fume hoods, respectively. Team members are trained in the proper use and precautions associated with various laboratory equipment and are educated on potential hazards and their mitigation. Any accidents or equipment malfunctions must be immediately reported to the advisor or safety officer.
We utilize three strains of E. coli in our experiments: DH5α, BL21 (DE3), and Nissle 1917. The DH5-alpha strain is used to amplify vectors containing biobricks for protein expression. The BL21 (DE3) strain is employed for expressing mGL (Green Fluorescent Protein), Zif268-TPH1 fusion protein, PBSII-PCBD fusion protein, and ZFa-QDPR fusion protein. These strains are classified as BSL-1, posing minimal risk to human health. Following biosafety guidelines, all biological waste is sterilized via autoclaving, and the lab environment is decontaminated using bleach or 75% ethanol.
EtBr is used in agarose gel electrophoresis to verify the correct size of gene blocks. As a potential mutagen, EtBr intercalates with double-stranded DNA and RNA, and excessive inhalation can be lethal. To minimize risk, we have designated a specific area for EtBr-related experiments. Gloves are worn at all times in this zone, and used EtBr is decontaminated through two days of sunlight exposure.
We designed a lethal genetic circuit to ensure that the genetically modified Nissle 1917 probiotics do not increase or affect non-target organisms in the environment. This circuit ensures that the probiotics are safely excreted from the human body. The circuit includes a TetR promoter and a TlpA promoter, functioning as a Tet-off system and a temperature-sensitive control, respectively. Additionally, we incorporated CRISPR/Cas9 technology, with cas9 and gRNA targeting SOS system-related genes in Nissle 1917. Cas9 and gRNA are regulated by the TetR promoter, which is controlled by the TlpA promoter. This design ensures that Nissle 1917 will trigger a suicide switch below 37°C (body temperature) but remain safe above this temperature.
During the development of SERENE, it is inevitable that certain chemicals with safety concerns will be used. However, strict safety protocols and innovative design strategies are in place to minimize risks and ensure the safe execution of all experimental procedures.