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Safety and Security

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Lab Safety

Prior to beginning laboratory work, our team was required to complete a series of training courses mandated by the Office of Environmental Health and Safety at our university. These online courses included the following:

In the weeks leading up to the start of our wet lab, all team members also underwent in-lab training under the guidance of our Principal Investigator, Dr. Keith Kozminski, and Lab Specialist Ms. Jackie Parker. We familiarized ourselves with lab equipment, potential hazards, waste disposal, safety protocols, general procedures relevant to our project, and eyewash stations and showers.

While working in the lab, all team members wore appropriate PPE and followed dress code guidelines, including tying back long hair and wearing closed-toe shoes. Upon completing experimental protocols, members followed proper lab hygiene procedures, including washing hands thoroughly with soap and water, disinfecting benches with CaviCide, and disposing of biological or liquid waste according to institutional health and safety standards.

Flasks and cuvettes containing cell media, cultures, or other biological materials were thoroughly bleached and then rinsed prior to reuse. Protocols involving hazardous chemicals, such as ninhydrin were performed under the fume hood to prevent respiratory irritation and other health risks. When handling toxic substances like propidium iodide, lab members wore double gloves to reduce exposure to carcinogens. To further improve safety, we substituted certain reagents such as replacing the mutagen ethidium bromide with the safer alternative Gel Red when casting gels.

Throughout the project, we received regular guidance on lab safety and protocols from our Principal Investigator Dr. Keith Kozminski and Lab Specialists Ms. Jackie Parker and Ms. Kay Christopher.

Chassis Safety

For our experiments, we selected non-pathogenic strains of E. coli K-12 (“BW25113”, JM109, MG1655, NEB 5-Alpha Turbos, Takara Stellar Competent Cells, One Shot® ccdB SurvivalTM 2 T1R), which are classified as Biosafety Level 1 (BSL-1). This classification indicates that the strains pose minimal risk and are not considered a significant threat to healthy individuals. Standard safety practices such as wearing gloves and lab coats are sufficient for handling BSL-1 organisms. We chose these strains to ensure our safety and that of others.

Kill Switch

Our long-term goal is to deploy our engineered chassis in dark fermentation bioreactors. To prevent our device from propagating outside of bioreactors, we decided to incorporate a kill switch based off the CcdA/CcdB toxin-antitoxin system, which is a well characterized Type II toxin-antitoxin mechanism native to E. coli K-12.

The CcdB toxin disrupts DNA replication by targeting DNA gyrase, a topoisomerase crucial to preventing excessive tangling during DNA replication and transcription in bacteria [1]. CcdB forms a covalent GyrA-DNA complex that leads to breakage of both plasmid and chromosomal DNA, resulting in eventual cell death. Ccdb activity is normally blocked by the CcdA antitoxin, which binds to CcdB and neutralizes its effects. In addition to neutralizing CcdB, CcdA plays a role in repressing the transcription of the ccdAB operon.

In our system, CcdA is constitutively expressed at a baseline level to mitigate any leaky CcdB expression. However, once extracellular cysteine crosses a threshold of 300 mg/L, CcdB expression is upregulated, eventually overwhelming the antitoxin and triggering cell death.

This kill switch acts as both a mechanism for our device function and a necessary biosafety layer, ensuring that our engineered cells do not propagate if taken outside their intended environment.