Organisms
The main organism used was Methylobacterium extorquens AM1 as it contains the natural lanthanide-binding protein lanmodulin and already contains lanthanide uptake and sensing machinery [1]. We also used different strains of Escherichia coli to generate stocks of our assemlbed plasmids (DH5α) and express our XoxF protein (BL21). These all have negligible individual and community risk as they are Risk Group 1 microorganisms belonging to the iGEM Safety White List.
Chemicals
| Chemical | Risk | Mitigation of Risk | Reference |
|---|---|---|---|
| 1X TAE buffer | May be harmful if inhaled, absorbed or swallowed. May cause eye irritation. Low risk. | Lab coat and gloves worn while handling. Awareness of first aid measures in case of emergency. | MCLAB. |
| 2,6-Dichlorophenolindophenol (DCPIP) | May skin irritation or eye irritation or be harmful if swallowed. Low risk. | Lab coat and gloves worn while handling. Awareness of first aid measures in case of emergency. | EDVOTEK. |
| Agarose | Low risk. | Lab coat and gloves worn whilst handling. | Fischer Scientific. |
| DTT | Harmful if swallowed. Could cause skin/eye irritation. Low risk. | Hands washed thoroughly after handling and protective equipment worn. Awareness of emergency procedures. | Cayman Chemicals. |
| EDTA | Harmful if inhaled. May cause organ damage through prolonged or repeated exposure if inhaled. Low risk. | Avoided breathing in dust. PPE worn and awareness of what to do in an emergency. | Sigma-Aldrich. |
| HCl, 6M | Skin corrosion and irritation, Eye damage, Respiratory system damage if inhaled. | PPE worn and awareness of what to do in an emergency. Stringently avoided breathing in vapour. Use minimised by preparing HCl-containing solutions in sufficient quantities for whole project exactly once. | ThermoFisher |
| IPTG | Can cause eye irritation and possible carcinogen. Low/medium risk. | Lab coat and gloves worn while handling. Awareness of first aid measures in case of emergency. | EDVOTEK. |
| LB broth | Low risk. | Lab coat and gloves worn whilst handling. | Fischer Scientific. |
| Methanol | Highly flammable, toxic if swallowed, in contact with skin or inhaled, health hazard. Low/medium risk. | Methanol was always handled in a fume cupboard and never near naked flames. It was also stored in a flammables cupboard. Lab coat and gloves were worn and skin contact was avoided. Awareness of first aid measures in case of emergency. | CLEAPPS. |
| Neodymium (III) chloride | May cause respiratory irritation. Low risk. | Lab coat and gloves were work whilst handling. Hands washed thoroughly after handling. Awareness of first aid measures in case of emergency. | Fischer Scientific. |
| Phenazine methosulfate (PMS) | Low risk. | PPE worn. Awareness of what to do in case of emergency. | Sigma-Aldrich |
| Pyrroloquinoline quinone | Can cause skin irritation, eye irritation and possible respiratory irritation. Harmful if swallowed. Low/medium risk. | Avoided inhaling the chemical. Lab coat and gloves work while handling. Awareness of first aid measures in case of emergency. | Cayman chemical |
| Tris buffer | May be harmful if inhaled, absorbed through skin/eyes or ingested. Low risk. | PPE worn. Awareness of procedures in case of emergency. | EDVOTEK. |
| Triton-X-100 | Acute oral toxicity. Can cause eye damage/eye irritation. Hazardous to aquatic life. | PPE was worn and release to the environment was avoided. Awareness of emergency procedure. | ROTH. |
| Urea | May cause eye, skin or respiratory system irritation. Low risk. | PPE worn. Awareness of emergency procedure. | Cayman Chemical. |
A choice that we made with chemical choice regarding safety is using protocols using DTT instead of beta-mercaptoethanol. Both are reducing agents however DTT is less toxic and less reactive so we chose to use this instead.
Lab Induction
All team members were required to complete and pass six courses before being allowed access to the lab. These include:
- Chemical Awareness
Control of substances hazardous to health, risk assessment, safety data sheets, chemical labelling, emergency procedures, general safety etc. - Fire Safety Training
Fire safety awareness, fire extinguisher and fire warden training. - Health and Safety Induction Training
Health and safety best practices, fire procedures, responsibility as staff etc. - Waste Management and Safety
Processes, procedures and decision-making in purchasing and using materials for the laboratory. - Sweep and tag system
The emergency evacuation process of the life sciences department at the university. - Local Exhaust Ventilation User Training
Structure and importance of LEV systems, their consequences, how they work and their limitations and what to do before use, during use and after use of them.
We worked in a biosafety level 1 standard microbiological laboratory. Areas used include an open bench to conduct our experiments, chemical fume cabinets for more hazardous chemicals and laminar flow hoods for maintaining a sterile environment when a flame wasn’t suitable; for example, using small aliquots of flammable chemicals such as methanol. Safety equipment available included eyewash stations, safety showers, fire extinguishers, first aid kits and fire blankets.
Lab Safety and Precautions
Precautions were followed as per the laboratory rules of the School of Life Sciences and Warwick Medical School such as:
- Lab coat worn and always fastened while working in the laboratory.
- Appropriate shoes worn covering the foot.
- Long hair tied back.
- Hands washed before leaving the laboratory.
- Awareness of control measures required by the risk assessment such as when to use fume cupboards.
- Awareness of what to do in case of emergency.
- Face, eyes or mouth not touched in the laboratory.
References
^[1] Mattocks, J.A., Jung, J.J. (2023), Lin, CY. et al. Enhanced rare-earth separation with a metal-sensitive lanmodulin dimer. Nature 618, 87–93