Every project, especially those involving biological lab work with potential future applications in a medical setting, carries inherent risks. Throughout the wet lab process, we have become more adept at performing laboratory techniques, working efficiently and effectively as a team, and maintaining a high standard of lab safety. We have taken great care to minimise risks, not only to comply with iGEM regulations (note: responsibility.igem.org. (n.d.). iGEM Responsibility. [online] Available at: https://responsibility.igem.org/safety-policies/introduction ) and local laws (note: Hse.gov.uk. (2020). What The Law Says about Biosafety. [online] Available at: https://www.hse.gov.uk/biosafety/law.htm#:~:text=The%20main%20piece%20of%20legislation%20that%20applies%20to%20infections%20at. ) but also to ensure our own safety and uphold ethical standards. Rigorous safety protocols have been applied to our laboratory practices and hardware, while also considering necessary safety measures for future implementation.
Wet Lab Experimentation
Escherichia coli DH5α refers to a specific strain of the bacterium Escherichia coli (E. coli), commonly used in molecular biology for cloning and transformation experiments. The DH5α strain is particularly favourable because it has genetic modifications that enhance plasmid uptake, making it "competent" for transformation, while also lacking certain endonucleases that would otherwise degrade introduced foreign DNA. (note: Kostylev, M., Otwell, A.E., Richardson, R.E. and Suzuki, Y. (2015). Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies. PLOS ONE, [online] 10(9), p.e0137466. doi:https://doi.org/10.1371/journal.pone.0137466 )
The DH5α strain of E. coli will be engineered to express ComC proteins along with GFP (Green Fluorescent Protein). ComC is a protein that is part of the natural competence system in some bacteria, which is involved in the uptake of extracellular DNA. However, in this context, the ComC protein is being studied for its potential role in increasing copper uptake in the engineered bacteria.
The GFP serves as a marker protein that fluoresces under UV light. This makes it easier for us to track and visualize the success of the transformation and gene expression in the bacteria. GFP acts as a reporter, allowing us to see whether the ComC gene and other introduced genetic elements are being properly expressed inside the E. coli DH5α cells. Two plasmids will be created through iGEM Biobrick assembly - the first carrying the ComC gene, the second coding for the GFP - and will then be transformed into two different sets of competent DH5α cells. (note: Chalfie, M. (1995). Green fluorescent protein. Photochemistry and photobiology, [online] 62(4), pp.651–6. doi:https://doi.org/10.1111/j.1751-1097.1995.tb08712.x. )
The synthesized part containing the ComR coding sequence from Top10 bacteria will serve as the insert for a plasmid, which will be transformed into Escherichia coli DH5α cells. The GFP part in this experiment contains a sequence from the ComC promoter region of Top10. This promoter includes the binding site for the ComR protein. (note: Thermofisher.com. (2024). TOP10 Competent Cells | Thermo Fisher Scientific - UK. [online] Available at: https://www.thermofisher.com/uk/en/home/life-science/cloning/competent-cells-for-transformation/competent-cells-strains/top10-competent-cells.html#:~:text=TOP10%20cells%20are%20the%20most%20popular%20E.%20coli%20strain%20engineered )
When ComR binds to the ComC promoter region, it regulates the expression of GFP, making fluorescence a measurable output. This GFP system allows us to then visualise and quantify gene expression based on fluorescence intensity, providing an indirect measure of how well ComR is functioning in the DH5α cells in different concentrations of copper.
Risks & Safety Measures
| Hazard | Risk | Safety Measure / Prevention |
|---|---|---|
| Exposure to E. coli (Non-pathogenic Strain DH5α) | Accidental exposure to the bacteria, such as through direct contact or inhalation, could lead to unintended infections, especially if basic hygiene and safety practices are not followed. Infection: While DH5α is non-pathogenic, exposure through cuts, open wounds, or mucous membranes (e.g., by touching one’s face, eyes, or mouth) could lead to localised infections, particularly in individuals with weakened immune systems. Environmental Contamination: Improper disposal of bacterial cultures or careless handling can lead to contamination of surfaces and equipment, increasing the risk of exposure to others in the laboratory. | Gloves: Wear gloves at all times when handling bacterial cultures to avoid direct contact. Face Masks and Goggles: To protect against potential splashes or aerosols, ensure the use of face masks and goggles when working with liquid cultures or during transformation experiments. Lab Coats: Wearing a lab coat minimises the risk of bacteria coming into contact with skin or clothing, and helps prevent the spread of bacteria outside of the lab. Avoid Touching Face: Refrain from touching face, mouth, eyes, or nose while working in the lab. Contaminated gloves or surfaces can transfer bacteria to mucous membranes, leading to infection. Hand Hygiene: Wash hands thoroughly with soap and water after handling bacterial cultures and before leaving the lab. Surface Disinfection: Regularly disinfect work surfaces, equipment, and tools with appropriate biocidal agents to prevent contamination and accidental spread of E. coli. Ensure that bacterial cultures and waste materials are disposed of properly in designated biohazard containers. Use biosafety cabinets when working with cultures to prevent airborne contamination. Autoclave all biohazardous waste to kill any live bacteria before disposal. |
| Exposure to Hazardous Cleaning Solutions | Many laboratory cleaning agents, such as bleach, ethanol, and other chemical disinfectants, can pose risks if not handled properly. These chemicals are often corrosive, irritating, or toxic if inhaled or exposed to skin, making proper handling and PPE essential. | Skin and Eye Irritation: Direct contact with these substances can cause chemical burns, irritation, or serious eye damage. PPE: Always wear gloves, goggles, and, if needed, face masks when handling cleaning chemicals. Inhalation: Some cleaning chemicals, like bleach or ethanol, release harmful vapours that can irritate the respiratory system, especially in poorly ventilated areas. Ventilation: Work in a well-ventilated area to avoid inhaling fumes. Use fume hoods when necessary. Chemical Reactions: Mixing certain cleaning agents (e.g., bleach and ammonia) can produce dangerous gases such as chloramine vapours, leading to respiratory distress. Labelling and Storage: Store cleaning agents properly and ensure they are clearly labelled to avoid accidental misuse or dangerous chemical reactions. |
| Working with Glass Equipment | Glassware such as beakers, flasks, and pipettes are commonly used in the lab but pose risks of injury due to their fragile and sharp nature. Breakage can lead to cuts, punctures, or even chemical exposure if the broken glass contains hazardous substances. | Cuts and Lacerations: Broken glass can cause deep cuts or punctures, leading to injury and possible contamination with chemicals or biological materials. Proper Handling: Always handle glassware with care, particularly when it contains liquids or chemicals. Avoid using cracked or damaged glassware to minimise the risk of breakage. Broken Glass Disposal: Immediately clean up broken glass with a brush and dustpan—never use your hands directly—and dispose of it in designated sharps containers. Be mindful of glass contaminated with chemicals, which may require specific disposal procedures. Chemical Exposure: If glassware containing hazardous chemicals breaks, it can result in accidental exposure through skin contact, inhalation, or ingestion. |
Application
We intend for the project to be introduced into hospitals to address nitrous oxide emissions from patients. The bacteria culture would act as a biofilter, aiming to break down this gas and reduce both staff exposure and its environmental impact. Having spoken to people working in the sector, we decided that the most sensible course of action would be to move the processing of the gas off-site in order to minimise the risks that come with exposure in a clinical setting. We also envision that kill switches, like the cold shock system, could be used to further reduce the risk.
Policies / Regulations
The Control of Substances Hazardous to Health Regulations 2002 https://www.legislation.gov.uk/uksi/2002/2677/contents/made and the Genetically Modified Organisms (Contained Use)
Regulations 2014 https://www.legislation.gov.uk/uksi/2014/1663/contents/made
At City of London School, lab safety is observed using CLEAPSS guidance, in addition to input from technicians where applicable: https://science.cleapss.org.uk/Resources/Student-Safety-Sheets/
KCL Health, Safety, Welfare and Fire Safety policy can be found here: https://www.kcl.ac.uk/policyhub/health-safety-welfare-policy. This page was provided to us by staff at KCL and was used to guide our lab work.
In the lab at King’s College London, we consulted Klaire Neale and Dr Anatoliy Markiv for practical guidance. For theoretical safety considerations and future implementation, we discussed requirements with Lyndsay Muirhead, which guided the prototype’s hardware design, including filters and UV light to prevent exposure.
KCL Lab Safety Record
We managed biocontainment risks by adhering to regulations and protocols from our training and King’s College supervisors. Key measures included proper waste disposal in autoclave bags and wearing gloves and goggles. KCL staff were always available for consultation. We were able to consult staff at KCL on any questions we had as they were constantly around.
We used a teaching lab in the Franklin Wilkins Building at KCL, equipped with gel electrophoresis tools, cloning equipment, an incubator, and freezers. The well-maintained equipment was regularly used for undergraduate teaching. We sought advice when using new equipment and did not require a biosafety cabinet for our protocols. Before entering and leaving the lab, we washed our hands at the provided sinks. We wore PPE, including gloves, lab coats, and goggles, when handling materials.
Waste was sorted into normal waste, gloves, sharps, and biological material. KCL technicians managed disposal, with biological material autoclaved in autoclave bags before disposal. Sharps were placed in bins emptied regularly to avoid overfilling, and bins were clearly marked to prevent improper disposal of gloves. After using bacteria on open benches, we cleaned the area with 1% Virkon.
No accidents occurred during the week. A KCL staff member, either Dr. Markiv or a technician, was always present to address any issues. Sharps and broken glass bins were available, and spills were cleaned with 1% Virkon. There was no fire risk as no flammable substances were used, but flammable solvents were stored in a designated cabinet with clear signage.
Before entering and leaving the lab, we washed our hands at the sinks provided. PPE including gloves, lab coats and goggles was provided and worn when handling materials.