Safety and Security
Safety and security in synthetic biology are crucial for preventing accidental harm to living creatures and the environment.
Work with any biological material requires special precautions. It is important to establish safety rules and educate team members about potential risks and ways to minimize danger.
Here you can take a look at how our team approached matters related to safety and security in our project.
Short introduction
Our project aims to develop toehold switches that may become a new treatment for hepatocellular cancer. We hope that toehold switches we designed will turn to a safe form of aimed cancer therapy. Our laboratory research involves work with cell cultures (human cell lines: HepG2, HEK293, HeLa, A549, WI38,), bacteria transformation (Escherichia coli bacteria strains: dh5alfa, dh10beta) and nucleic acid isolation. Some of the techniques used during the project include substances that may cause threat to human health. That is why it was important to establish safety rules and educate our team members about potential risks and ways to minimize danger.
Lab safety
At the beginning, we had a week-long training camp for all members of our Wet Lab iGEM's team. During the first half of the event we completed the following safety courses:
- laboratory safety training
- fire safety and evacuation
- occupational health and safety orientation
- biosafety training
- spill response training
- hazard assessment training
- cyber security
The second half of our training camp covered the practice of laboratory techniques that will be used during our iGEM project. During that time we got familiar with the laboratory space and equipment.
Lab equipment
Our laboratory has restricted access, so no-one without a pass card can invade our work space that holds possibly dangerous biological material and chemical reagents. A well equipped First Aid Kit is present and visible from every place in the lab. There is an emergency wash station (visible from every place in the lab) that all team members were instructed on how to use.
The lab bench is made of a stain resistant, chemical resistant and fireproof material. There are disinfectants and cleaning tools present on each bench, which we use before and after each experiment to prevent contaminations and uncontrollable spreading of biological material or chemicals. We keep organised pipettes, gloves, plastics and kits for DNA and RNA isolation (only with safe reagents) above the bench. All reagents are labeled and organised in the lab space, stored in their designated temperature (in cabinets of room temperature, fridges +4°C or freezers -20°C & -80°C). Moreover, dangerous reagents are additionally labeled with danger risk signs and stored in a separate cabinet and laboratory hood. There are separate rooms for cell culture and bacteria culture to prevent contamination and uncontrollable spreading of biological material.
Bacteria culture room
The bacteria culture room has its own: incubator, fridge, autoclave, fire work station, microwave, red bin for laboratory waste, disinfectants and cleaning tools, centrifuge, electrophoresis tool.
Cell culture room
The cell culture room has its own: laminar flow hood with UV light sterilization, incubators, fridge, microscope, red bin for laboratory waste, disinfectants and cleaning tools.
Waste disposal
Our team segregates waste that has not had contact with any biological material (mostly plastic or paper/cardboard packaging) in standard segregation bins. The special red bins are used for every piece of waste that had contact with chemicals and biological material such as those autoclaved, affected by treatment with bleach and ethanol, or if they are to be collected by a special company.
Personal safety
While working in the lab, every team member needs to wear Personal Protective Equipment (PPE), which includes: gloves, lab coat, closed-toed shoes, long hair tied back, eye protection (glasses or goggles) if working with more dangerous substances or equipment. It is expected that they will disinfect and wash their hands after entering the laboratory and before leaving the lab.
We make sure that each member of the team is vaccinated for HAV and HBV for safety reasons in case of accidental cuts in the laboratory space. As in many laboratories we use some hazardous chemicals in our project for example ethanol (which is highly flammable) or acrylamide and TEMED. We also decided to switch (if possible) from harmful substances like ethidium bromide to safer substitutes like RedSafe. For our reagents, we cataloged and arranged proper storage. Similarly, every dangerous chemical is thoroughly labelled. Additionally, we care for our team members’ wellbeing, so we make sure that there is a resting space inside the building for eating and resting outside the laboratory space.
Biological safety
Work with any biological material requires special precautions. Members of our team need to follow cleaning procedures before and after conducting experiments. It is expected that they will disinfect and wash their hands after entering the laboratory and before leaving the lab. It is also important to disinfect the lab bench before and after experiments to prevent cross contamination of samples. We know that every biological material designed in the laboratory should not leave the lab space uncontrollably. It might otherwise cause a threat to the environment due to its own nature or its subtle impact on the surrounding.
Toehold switch
Our construct is based on RNA technology, similar to the well known, safe and tested vaccine mRNA technology. The main difference between our toehold switches and the vaccine mRNA is that our construct has a hairpin structure. It needs to be opened by a compatible mRNA sequence to achieve an active state, unlike typical mRNA which is already in an active - ready to read by ribosome, state. Our toehold switches involve a fragment that detects mRNA that codes alfafetoprotein, which for adult humans presents only in hepatocellular cancer. Although there is little chance of off-target reactions, we still tested our construct in this direction on different cell lines. The product of expression of our toehold switches is gasdermin, which is a protein naturally occurring in the human body and is known to be a tumor suppressor. Gasdermin also takes part in cell signaling, in which it triggers pyroptosis - a cell death way that occurs naturally in the human body and involves an immunological response. Due to this fact, later, when aimed cancer therapy will be established, smaller amounts of therapeutics could be used to achieve tumor destruction. Although all from above is general safety of our construct, much more testing should be performed before clinical tests or usage.
Bacterial strains
Escherichia coli is usually a harmless bacteria, commonly used in laboratory practice. Although we are using safe strains that are not producing toxins, we keep all safety measures while working with E. coli strains.
E. coli strains dh5alfa and dh10beta are chemocompetent bacteria (and one of the most common laboratory strains), especially altered for general cloning and high transformation efficiency. We keep in mind that every genetically modified organism may pose a threat to the environment, so we take all safety measures while working with these bacteria and during their disposal.
All dishes and flasks with bacteria culture are kept in a designated incubator. To prevent contamination of bacteria culture, we first disinfect the bench and later (after ethanol completely evaporated) work in a sterile environment (15 cm radius) around a flame. While working with a flame, we have to remain especially cautious and keep flammable materials away, having a source of water and a fire extinguisher nearby.
All plastics that were in contact with bacteria are disposed of into the red bin. A one-way liquid flow is kept while working with bacteria and any waste liquids are sterilized before disposal.
Safety measures
- PPE: Lab coats, gloves, and goggles. Closed-toed shoes and long sleeves required.
- Emergency Preparedness: Eyewash stations and emergency kits are accessible.
- Cleaning: Benches disinfected before and after work.
- Work Area Designation: Operations only in designated benches, no lab-to-lab travel with materials.
- Sterilization: Regular ethanol/bleach cleaning.
- Autoclaving: All glassware and plastics autoclaved before disposal.
- Waste Management: Red bins and labeled containers used for biohazardous materials.
Human cell lines
All human cell lines are considered possibly hazardous and classified as Biological Safety Level 2. This is why we work under a laminar flow hood while wearing gloves and a lab coat. For our own safety, we make sure that all cell lines are free from viruses (HPV, HIV) and mycoplasma (tested negative). All waste that had contact with cells goes to the red bin and is later collected and safely disposed of by a specialized company.
Human cell cultures are fragile and prone to contamination. That is why we frequently disinfect the work area, turn on UV light in the laminar for 10 minutes before and for 30 minutes after each use, and disinfect all items with ethanol before placing them in the hood. Dishes with cells are kept in a sterilized incubator, and their exposure outside is minimized.
Experiments such as transfection, RNA isolation, or mRNA construct assay are performed only in designated benches and laminar flow hoods. All plastics that were in contact with cells are disposed into red bins. Waste liquids are sterilized before disposal.
Safety measures
- PPE: Lab coats, gloves, and goggles. Closed-toed shoes and long sleeves required.
- Emergency Preparedness: Eyewash stations and emergency kits are accessible.
- Cleaning: Benches disinfected, UV sterilization before/after work.
- Work Area Designation: Operations only in laminar hood and designated benches.
- Sterilization: Ethanol, bleach, and UV cleaning protocols followed.
- Autoclaving: All tools, plastics, and glassware autoclaved before disposal.
- Waste Management: Red bins for biohazards, broken glass protocols followed.
Risk management and STPA
Hazard analysis is an important step when designing any synthetic biology project. While there are undeniable benefits, products of genetic engineering may pose risks. We used System Theoretic Process Analysis (STPA), an iterative hazard analysis tool developed by MIT’s Nancy Leveson, to identify threats and mitigation strategies.
This method, already used in aviation, nuclear plants, and recently in iGEM (UCalgary 2024 team ErythrO2), proved effective in synthetic biology as well. We developed simple short guide as a complementary material for performing STPA, which we love to share for future teams.
Download our short guide!
Why STPA and not other systems?
Unlike linear hazard analysis (FTA, ETA, FMECA, HAZOP), STPA does not assume accidents only come from component f ailure. Instead, it considers unsafe interactions and lack of controls. This makes it more suitable for biological systems with complex networks of interactions.
References:
John Thomas, "Systems Theoretic Process Analysis (STPA) Tutorial," MIT, 2013, © Copyright John Thomas 2013 [PDF]
Leveson, N. G., & Thomas, J. P. (2018). STPA handbook. [PDF]
Leveson, N. G. (2023). An introduction to system safety engineering. MIT Press.
2024 iGEM UCalgary team ErythrO2: [Link]
Cyber security
Naturally, with any modern project, electronics become involved with the processes, be it communication, sharing data or storing backup results. We have to make sure to assess cyber security properly and make some arrangements in this area of our project. Firstly, we agreed that all team members will use emails from the same domain. Then we established a few communication channels (messages chats and phone calls for topics that require quick answers, slack chats for more official communication, emails for official outside communication and goggle meet for online meetings). We decided to keep our lab book notes and protocols on benchling accounts and for all files there is backup on our google drive, same goes for the results of the experiments.
As for the bioinformatics part, we ensure the use of up-to-date libraries and frameworks in our code. We create backups and ensure confidential data is not disclosed. On our educational platform, we control user access to data at the application level with role-based permissions. We ensure GDPR compliance by minimizing the amount of collected personal data, storing only necessary information, and hashing all passwords.
Our team plans to utilize artificial intelligence (AI) tools, which naturally carries certain risks. One of the main ones is the risk of inaccurate or biased predictions, especially in genetic target identification, which could lead to ineffective or non-specific toehold designs. To mitigate this, we will validate all AI-generated outputs with established bioinformatics pipelines and manual expert review.
Another risk is overreliance on AI-driven experimental design, which might overlook biological nuances. We will address this by cross-checking AI-designed experiments with current literature and consulting experienced researchers before implementation.
In data analysis, AI may misinterpret patterns due to noise or technical artifacts. We will apply rigorous statistical controls and use multiple analytical methods to ensure robust results.