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How We Practiced Safety
As we were working with perfluorooctanoic acid (PFOA), a known carcinogen that causes harm in humans and the environment by accumulation, we mitigated the risk by only allowing trained adult personnel to handle the PFOA. They followed the Department of Environmental Health and Safety guidelines and approved Standard Operating Procedures (SOP). Any waste from the PFOA was collected and disposed of by the University of Louisville's Department of Environmental Health and Safety.
Our project would provide a cost-efficient and user-friendly device that would allow people to determine the amount of PFAS in their liquid sample. The solution will be safe for the user as well as the environment. The future development of this project would not require release beyond containment. Before taking our project outside of the lab, we would utilize auxotrophy in our containment device so that, should any cells escape, they could not survive in the environment.
To ensure the new plasmid is safe, cautious checks were carried out via the Synthclient system of the SecureDNA platform. The plasmid was checked by submitting a FASTA file, a standard method of presenting nucleotide sequence information. SecureDNA holds the information confidential and secret, meaning the actual sequence cannot be viewed during checking. Instead, the vector is converted into hashed sequences, allowing for increased privacy without compromising significant genetic information.
Our screening results showed that our plasmid sequence contained no toxic or unlawful molecules through the JSON script "synthesis\_permission: granted," from SecureDNA. Our verification also demonstrates that we are adhering to the safety guidelines while establishing credibility concerning the efficacy and synthesis capabilities of our vector sequences.
We conducted our wet lab work at the University of Louisville's Genomic Training Facility Center under Sabine Waigel and Kaline Andreeva-Stallard, and the Brown Cancer Center. All work conducted in the lab during our time there was compliant with the United States' lab safety regulations. We ensured that all activities strictly abided by these regulations. All handling of PFOA will be done by double-gloving under a fume hood by an adult due to its safety concerns. Otherwise, we work in an open lab bench. If anything were to happen, we would contact our EHS Training Specialist and IBC/Lab Safety Coordinator.
To ensure project-specific safety, we developed an SOP for handling PFOA. After consulting with other experts about managing the risks of PFOA, we modified our IBC protocol to include the BL21 strain. This modification means we did not use the original organism of the PrmA promoter (Rhodococcus jostii) and instead used E. coli.
The actions we have taken for waste disposal helped manage the risks in our project because we have been made aware of the issues that occur if we do not follow certain rules such as disposing of waste properly in 32-gallon red containers lined with biohazard bags, and not complying with the NIH guidelines, CDC recommendations, OSHA regulations, DEHS procedures, IBC guidelines, and EHS training. All PFOA-containing waste was collected in an appropriately labeled container and disposed of by DEHS at the University of Louisville. All E. coli-containing waste will either be treated with 10% bleach or, in the case of solid waste, will be collected with biohazardous waste and autoclaved.
For the first prong of our project, we transformed our plasmids into BL21 E. Coli, and then exposed E. Coli cells to PFAS, and finally, measured fluorescence of the cells. The plasmid for this project was ordered from GenScript, which is part of IGSC.
The second route used a cell-free system, where we expressed a protein that binds to PFAO. For this second prong, we transformed a plasmid to express our fusion protein in BL21 E. Coli. The plasmid for this experiment was ordered by the University of Louisville Protein Expression and Purification Core from VectorBuilder (Chicago, IL, USA). This company is not part of IGSC, but requires the customer to confirm the biosafety of their requested sequences. To ensure compliance with the iGEM biosafety review, the team screened the plasmid sequences themselves, using SecureDNA (SecureDNA Foundation, Basel, Switzerland).
The lab is classified as BSL-4 according to the Occupational Safety and Health Administration (OSHA) in the United States. All participants involved in wet lab work underwent the required training. The training was conducted by the University of Louisville's Department of Environmental Health and Safety representatives.
During training, we learned about waste management and what to do in emergencies. Since the Bloodborne Pathogens Program requires departments to make an exposure control plan, offer hepatitis B vaccinations, provide initial and annual training, and maintain records, we abided by these as well. The Institutional Biosafety Committee reviews activities involving biohazardous agents and recombinant/synthetic nucleic acid molecules and ensures compliance with NIH Guidelines, CDC recommendations, and OSHA regulations. Employees were also required to complete all EHS training appropriate for their work environment, such as General Lab Safety & Hazardous Waste, Basic Biosafety, Bloodborne Pathogens, Radiation Safety, IBC and NIH Guidelines, and Shipping Biologicals and Dry Ice.
We ensured to use personal and protective equipment (PPE) at all times. This included lab coats, gloves, and goggles to minimize exposure to potential contaminants.
Additionally, we used a Qubit Fluorometric Quantification for a quality assessment of the bacterial RNA samples. Some Qubit reagents are organic dyes provided in a solvent such as DMSO; therefore, we made sure to use proper PPE. Additionally, we worked in a well-ventilated area and followed proper disposal rules as administered by the DEHS of the University of Louisville.
We used a Bioanalyzer and consulted its Safety Data Sheet to ensure that we were following all safety precautions necessary. We used this instrument since it helped us with our sequencing libraries and the preparations for them.
All hazardous chemicals that we used were registered with the University of Louisville's Department of Environmental Health and Safety. Our lab safety training covered how we would act if ever exposed to PFOA, a carcinogen and corrosive chemical that would lead to several health effects. However, despite this, only adult-trained personnel were able to test the PFOA to prevent anything from happening to the children.
All experiments with the PFOA were completed directly under a chemical fume hood by trained personnel to abide by safety regulations.
As part of the project, we used AI through large language models (LLMs), protein structure prediction tools, and NeuralPlexer - an AI model that predicts protein binding sites for ligands. Because of this, we were faced with the risk of proprietary information being leaked before we were able to publish it. Because we verify information given to us by Large Language Models (LLMs) by researching peer-reviewed papers, false information is not a risk when using LLMs.