LAB SAFETY

Prior to any lab work, all members (both dry and wet lab) completed online lab safety training. Each course included a final examination which all members were required to pass to complete the training and receive a certificate of completion.

These courses include:

• Biosafety (Bloodborne Pathogens) Training
• Biosafety (Program) Training
• Fire Safety
• Spill Response Training
• Laboratory Safety Training
• WHMIS 2015
• Hazard Assessment Training
• Occupational Health and Safety Orientation
• Harassment and Violence Awareness Training
• Incident Reporting and Investigation Training
• LSIH Safety Training (not required for university lab work but completed for the MindFuel pitch competition)

Additionally, all members took an Introduction to Synthetic Biology course (MDSC 507) where we had lectures teaching us common synthetic biology laboratory techniques as well as wet lab training to practice various experimental procedures relevant to the development of our project. These training sessions familiarized us with lab equipment and their potential hazards associated with their use so we can be attentive and alert to minimize their risks. All lab training was conducted under the supervision of our principal investigators (Dr. Mark Ungrin and Dr. Xiaofan Jin), teaching assistants (Muskaan Puri and Dhruvi Kakadiya), and mentors from past UCalgary iGEM teams.

This year, our team worked in three different laboratory spaces: one chemistry lab and two biology labs. All spaces required different training and orientation before conducting any experiments in the lab. They all have very different safety and experimental protocols because of their different features and BSL levels.

Biology Lab #1 (BSL-2):

Orientation and Training:
Prior to getting lab orientation for this lab, members must complete all online safety modules listed above and attend an in-person orientation. Additionally, in order to use any lab equipment, an orientation by the lab technician must be performed first, then each member must show the lab technician they are capable of using the equipment by repeating the procedure independently under her witness. These lab equipment orientations were documented by filling out a Laboratory Health and Safety Orientation and Training Record form [1].

Waste Disposal:
Any item that came in contact with E. coli was disposed into the yellow biohazard waste bin. This includes petri dishes, and other solutions containing the bacteria. Every other item that was not contaminated with E. coli was placed in waste containers on top of the lab benches. Items such as pipette tips are brought to autoclave.

Experiments Conducted:
This lab was originally designed for biomedical engineering applications so it houses a wide range of instruments. However, it did not have the necessary equipment for synthetic biology experiments such as a thermocycler. No chemistry experiments were conducted in this lab due to the absence of aqueous and organic waste disposal containers. Since this lab is BSL-2 certified, it was suitable to handle bacteria which allowed us to carry out work on the phagemid during early stages of project development.

Biology Lab #2:

Orientation and Training:
This laboratory belongs to our PI, Dr. Mark Ungrin. Training for this lab included an in-person lab orientation given by the two graduate students, one of which is our TA, in the Ungrin lab, which followed the same criteria from the Laboratory Health and Safety Orientation and Training Record [1]. In early stages of working in this lab, members required TA supervision to conduct experiments. Members gained independent lab access once they proved their responsibility to work in the lab space unsupervised.

Waste Disposal:
There was a variety of specific waste containers in this laboratory:

• Biohazard “sharps”: This includes pipette and needle waste
• Biohazard solid: Any other solid material that came in contact with bacteria
• Mercaptoethanol: There were waste containers in the fume hood for specific chemicals, one being mercaptoethanol
• Ethidium bromide: This was another specific chemical waste container in the fume hood
• Hazardous liquid: General hazardous waste was for any other liquid chemical waste that did not have a specified waste container in the fume hood
• Gel waste: Any gel that was run (agarose or polyacrylamide) was placed into this solid waste container
• General: Any consumables that are not contaminated with bacteria or chemicals

Any liquid bacteria waste was treated with 70% bleach and poured down the drain.

Experiments Conducted:
This lab contains the most extensive range of instruments as it belongs to our PI Dr. Mark Ungrin. Once access to this lab was granted, all bacteria-related experiments were conducted here. While it is also equipped to perform chemistry-based experiments, the lab is located on a different campus. Therefore, to minimize disruption and out of respect for the Ungrin lab members, only bacteria-based experiments were performed in this space.

Chemistry Lab (no BSL certification):

Orientation and Training:
To begin working in this lab, members must complete standard safety courses for university level chemistry courses: WHMIS 2015 and Chemistry Undergraduate Online Safety Course, as well as an orientation of the laboratory. Before using an instrument that no member has used before, an orientation of it must be done by the lab supervisor. In the early stages of working in this lab, a TA must be present at all times for supervision. Once we proved to the lab supervisor we were capable of working unsupervised, he gave us permission to work independently under four conditions that must be followed every day we were in this lab:

1. Our wet lab lead must be present at all times
2. At least 3 members (including the wet lab lead) must be present in the lab
3. An email to the lab supervisor at the end of the day describing what we did in the lab
4. All university lab safety policies must be followed

Waste Disposal:
All chemical waste was disposed into its respective containers (ie. aqueous and organic). All solid waste was disposed of into the solid waste bin. Any waste containers were kept in a fume hood to minimize exposure of chemical vapours. Lab technicians must submit a catalog of the waste each month for proper disposal. We were required to document all solid, organic, and aqueous waste created into a waste log so the lab technician could submit the list to the university for hazardous waste disposal.

Experiments Conducted: Since this lab is a chemistry lab, it does not have any BSL certification. This considered, absolutely no E.coli was handled in this laboratory. Although no bacteria was prohibited in this lab, other biological molecules (eg. DNA) were permitted. Any chemistry-based experiment and experiments requiring DNA were conducted in this lab.

When working in all laboratories all lab safety protocols were followed. All members wore appropriate PPE (ie. lab coat, goggles, gloves) and dressed according to standard protocol (ie. long hair tied back, closed toe shoes, long pants with no rips, no hats obstructing vision, no footwear with unstable heels) [2]. Lab etiquette of members was expected the same as researchers in a lab setting. This included no eating or drinking in the lab, washing hands regularly during experimentation and before leaving the lab, no re-using gloves to prevent cross contamination, sanitizing work benches before and after use, maintaining an organized and clean work station, and disposing all solid and liquid waste in their proper container before leaving the lab. If waste needed to be cataloged, this was completed either during an experiment or before leaving the lab.

Besides maintaining proper lab hygiene and manners, members also chose to substitute certain chemicals for a safer, yet equally effective alternative. This includes using ethanol to rinse glass surfaces instead of strong acids such as aqua regia, and replacing ethidium bromide with a less carcinogenic alternative, SYBR Safe. All experiments in the chemistry lab were conducted inside a fume hood, regardless whether protocol deemed it to be necessary. This precaution was taken to prevent inhalation of toxic fumes and minimize exposure of chemical vapours.

All laboratories have standard emergency safety features such as eye wash stations, safety showers, fire extinguishers, and first aid kit. All three labs maintain additional lab coats and safety goggles to uphold dress code protocols in the event a member forgets their mandatory PPE. Excluding fume hoods, all labs have general room ventilation to prevent fugitive gas emission from building up [3].

RISK MANAGEMENT

Near-miss Analysis

The term incident is “an occurrence arising out of, or in the course of, work that could or does result in harm.” [1]. A near-miss, which is an incident that does not result in harm, but has the potential to, falls under this term. Near-misses are arguably the most common unplanned safety event that occurs in the workplace, however it is also the one that is the most overlooked. For this reason, the focus of this incident analysis will be put onto near-misses in the lab.

A near-miss differs from an accident because near-misses only have the potential to cause harm, but no harm actually occurred. In contrast, an accident is “an inadvertent occurrence that results in actual harm such as infection, illness, injury in humans or contamination of the environment.” [2]. We chose this definition of an accident because it encompasses both harm to humans, and the environment. Our project has a strong focus on agricultural safety, and one of our future directions is to advance our project to continuous environmental sensors. That being said, it is important for us to acknowledge the environmental aspects of lab safety as well.

Based on this definition, near-misses occur very frequently, especially in a laboratory setting. After a meeting with laboratory safety specialist from the Veterinary Medicine department of the University of Calgary, Dr. Janina Willkomm, it was learned that despite the regular occurrence of this near-misses, it still remains as one of the least reported safety events. Part of the reason for under reporting is due to the burden many individuals view safety reporting as. In science, many students, supervisors, and even principal investigators find safety reporting to be an inconvenience as it takes away from their time and freedom to do research.

At the University of Calgary, near-misses are reported in the same form as major incidents called the Online Accident Reporting System (OARS). This is a very long and insightful form that goes through the Environment, Health and Safety department at UCalgary. Many individuals do not want to go through the inconvenience of reporting an event that did not cause any harm in a 4-6 page long form. This results in only the major accidents to be reported, while the near-misses often get forgotten or ignored.

The goal of this analysis is to identify common incidents in the laboratory and develop mitigation strategies, preventative measures, and other corrective actions to minimize the risks of the associated hazards. The corrective actions determined in this analysis will be incorporated into future lab training for more UCalgary iGEM teams with the aim of fostering a stronger safety culture and ensuring a safer work environment. These controls can also be used for other iGEM teams as well because the controls are applicable to any laboratory, whether they are biology or chemistry labs.

We also wanted to begin developing a safety reporting system that makes safety reporting more accessible, and less of an inconvenience for researchers. Due to the low reporting outcomes of near-misses it can be difficult to develop training, mitigation strategies, and barriers to relieve incidents. By developing an easy and low effort form, it could help with reporting numbers, and foster a stronger safety culture within our iGEM team. This year, we mainly focused on the analysis portion because we soon realized after giving it careful consideration, that making a well thought out and effective reporting system can take a long time that requires multiple individuals to hone in on. Giving the size of our team, time for project development, and other responsibilities as a member of iGEM, we decided that making a thorough analysis that includes infographics as visual summaries will be best for this year. However, as a future direction, a safety reporting system is a notable element to work on along with the analysis.

After meeting with Denise Howitt, a senior manager from the Environmental, Health, and Safety department of the University of Calgary, we learned that there is no prescribed methodology that must be followed for near-miss analysis reports. This considered, we have created our own method of analyzing near-misses, which included the main points we wanted to focus on, causal factors and barriers/controls.

Parts of the Analysis

Category

We have categorized all of the incidents into six separate groups:

• Broken Glass: Any incident resulting in glass to be (or almost be) chipped, shattered, or cracked
• Chemical/Material Spill: Any incident resulting in chemicals or other laboratory materials to be (or almost be) spilled or leaked out of a container (glass or plastic).
• Chemical Reaction: Any incident resulting from multiple substances to be mixed together
• Trips/Falls/Slips: Any incident causing (or almost) an individual to physically lose balance
• Instrument related: Any incident related to instruments, equipment, or technology in the lab
• Needles: Any incident associated with needles/syringes or other sharp pointed ends

Besides categories for incidents, the incident will also be categorized into a near-miss, or accident because aside from near-misses, accidents were also documented to identify any more serious risks, and trends/relationships between incidents.

The above categories were chosen based on the cause of the incident. It is understood that there are many different ways an incident can occur. We compiled all of the incidents reported in our lab spaces and analyzed their causes. From there we came up with seven different categories. When choosing categories, there was no limit on how many categories there could be. We wanted to make the categories more general so they could encompass a vast range of incidents without going into their technical details (this would be performed in the analysis). The above categories are the ones for the first and current near-miss analysis performed at UCalgary iGEM, however it is worth noting that these categories could change, increase or decrease in number as more analyses are made and more incidents are reported. Nothing in safety is set in stone because safety culture, technology, and research are constantly developing.

Likelihood, Severity, and Risk Category

In this analysis we will be ranking the severity of the near-miss and likelihood of the near-miss to occur.

Likelihood is the probability of the incident to occur. There are two ways to rank the likelihood of an incident. The first being ranking the incident raw with no barriers in consideration. The second way is to rank them with the barriers in mind. The barriers/controls will be discussed in this analysis in the Barriers/Controls section, however we will be ranking the raw near-miss data (i.e. without considering the barriers).

The severity of near-miss is ranked based on how severe the consequence would be if control of the near-miss is lost. For example, a gas valve in the lab was left on after an experiment while everyone was cleaning up, but someone ended up noticing and closed in before everyone left the laboratory. No harm occurred and someone ended up relieving the situation, however if the gas valve were to be left open overnight, the next person using the lab would inhale all of the gas. If they were to use a bunsen burner, then a large flame would be ignited. The severity of this near-miss would be ranked 'high' because the consequences can result in toxic release of a chemical. As for accidents, the severity was ranked based on the resulting consequence.

Severity and likelihood will always be ranked a higher risk if the incident falls under multiple rankings. For example, if an incident results in a medical aid injury, and high repair costs, the severity will be ranked 'high' because it is always better to expect more severe outcomes so preparation can be made.

The following table was used to rank the severity and likelihood of each incident. This table was originally introduced to us by Lucca C. Filippo during the Risk Management Workshop he hosted for us. The severity ranking is followed by the acronym PEAPr, which stands for people, environment, assets, and production:

• People: Any harm resulting in injuries, illnesses, or other health related damages to humans
• Environment: Any harm done to the environment
• Assets: Any harm resulting in damage to equipment or facility that hinders financial stability
• Production: Any harm that results in interruption or delay of a projects progress

We have modified this table to be more applicable and feasible for an iGEM team. Main differences include the production section and likelihood column. Due to the limited funding for iGEM teams, the repair cost thresholds are significantly lower as even a $1,000 expense can have a substantial impact on an iGEM team's financial situation. Since most iGEM projects are over a 4 month period, the occurrence of an incident has been measured within a 4 month period. We also considered that all members are undergraduate students with limited self-run lab experience, so it is expected that more incidents may happen out of inexperience.

Table 1. Risk assessment table outlining how to determine the likelihood and severity of an incident. Modified from the table in the iGEM Risk Management Workshop hosted by Lucca C. Filippo to be more applicable to iGEM.

Ranking	Severity	Likelihood
High	P: Disabling injury, fatality E: Toxic release of chemical A: High repair cost (>$1K) Pr: Delay for extended period (>1 week)	Repetitive event Occurs several times (8≤ times in 4 months) Observed often in similar circumstances, but also occurs frequently in different ones
Medium	P: Medical Aid Injury E: Reportable spill, but non-toxic release A: Moderate repair cost ($500–$1K) Pr: Short delay (>1 day)	Infrequent event Occurs occasionally (4–7 times in 4 months) Observed occasionally in similar circumstances, rarely in different ones
Low	P: First Aid Injury E: No environmental damage and/or non-toxic release A: Low repair cost (≤$500) Pr: Brief interruption (≤1 day)	Unlikely event Occurs rarely (1–3 times in 4 months) Observed in a specific scenario

The risk category of a near-miss is determined by a combination of the likelihood and severity using the risk assessment matrix. The risk assessment matrix is a visual tool used to evaluate potential risks in a system. The combination of the likelihood and severity rankings is what determines the final risk category. The risk category of an incident then determines the urgency of required barriers and controls.

Causal Factor(s)

In simple words the causal factor is how the incident happened. It is what an individual was doing leading up to the event. Many different causal factors can lead to one incident, and at the same time an incident can be caused by many different scenarios. If many similar causal factors lead to a similar outcome, they will be grouped together for one analysis.

The causal factor was reported using a google form to collect near-miss data

Barriers/Controls

Similar to the bowtie analysis, there will be controls/barriers outlined for each near-miss to either prevent them from happening, reduce the likelihood of it happening, or reduce the losses of the consequence of the near-miss (ie. severity). One barrier could control many different incidents, so it will not be uncommon for barriers to be repeated in this analysis. At the same time barriers are usually quite simple and achievable. Most barriers outlined in this analysis likely will be actions that seem like “common sense.” However, in reality many individuals forget about simple actions that can be performed to easily mitigate the risk. The goal is to have easy, achievable and feasible barriers so they can be quickly and effectively implemented. These barriers are also ones that can be mentioned or emphasized on during lab training.

Infographics

As said before, safety has been viewed as a burden. As part to mitigate this, we have created infographics summarizing each category of incidents. Since the near-miss analysis is a long document, we understand that the length can be a factor causing hesitation to read it. With this information in mind and after a meeting with Denise Howitt, Denise gave us the idea to create infographics as a visual for others to view. Creating these infographics are similar to why we chose the bow tie method as our hazard analysis. It gives a visual representation of the information we want to convey without losing any key information. These infographics act as a summary to the whole analysis, but in a more eye pleasing way. These visuals can also be downloaded and printed out to keep in other laboratories or classrooms as safety reminders, and also to help with training future researchers.

Risk Management Workshop

As mentioned with the near-miss analysis, a focus of safety this year has been risk management which includes implementing controls and barriers to eliminate or reduce the impact of risks in the laboratory. This year, we were lucky enough to have Lucca C. Filippo, a graduate student from the University of Alberta's Lynch School of Engineering Safety and Risk Management, come in and host a workshop to teach us the importance of safety and risk management, how to assess laboratory hazards, and come up with effective barriers for risks.

The workshop consisted of four main activities:

1. Identify hazards in a laboratory
2. Assess the risks associated to the hazards identified in activity #1
3. Propose further measures to lower the risks of the hazards
4. Analyze the barriers proposed and answer the question: How safe is this system really?

In activity #1, we first familiarized ourselves with the preliminary hazard assessment (PHA) form. We learned that this assessment is a safety and risk assessment that is done early on in project development to identify potential hazards, consequences, and potential control measures. In the case of iGEM, it would be done before conducting any experiments in the lab so we can be proactive and prepared when it comes to hazard identification and best safety practices in the lab. We went to one of our lab spaces (biology lab #1) to analyze certain hazards in that lab as an exercise to complete the PHA. When identifying hazards, many of our team members mistaken consequences for hazards. An example would be saying a hazard is chemical burns from making a buffer, however in reality, a chemical burn is the result of being exposed to a chemical. Through Lucca's guidance, we learned that hazards are usually more generalized. So instead of saying chemical burn as the hazard, the hazard would actually be exposure of hazardous chemicals. At the same time, this activity taught us that a consequence is the result or outcome of a risk event.

For activity #2, we first proposed consequences for each of the hazards identified from activity #1, and then proposed barriers to prevent the consequences from occurring. We were also introduced to the concept of likelihood and severity of a consequence. We were introduced to the PEAPr acronym to determine severity and also a risk category. For all of the consequences proposed, we were tasked to give it a severity and likelihood following the PEAPr acronym for severity and the criteria in the table (given as reference material in the workshop) for likelihood. We also learned there are two different ways of determining severity and likelihood for consequences. One being assessing the raw risk without considering any barriers, and the other being assessing them prior to implementing the barriers. The risk category will likely be different depending on which route was taken.

Many parts of the near-miss analysis was chosen to be included after doing this activity. Prior to this workshop many parts of the analysis were not within our knowledge of safety and risk management, so this workshop has allowed us to further develop many parts of our safety plan. We thought everything we learned in this activity was very insightful to analyze risks in the lab so we decided to include it in our near-miss analysis.

In activity #3, we got introduced to the hierarchy of controls which helped us gain more insight on barriers and the effectiveness of different types of barriers. We learned that elimination and substitution barriers are the most effective. However, in science it can be very difficult to eliminate risks because nearly everything we handle and produce possess risks. That being said, most of our barriers were engineering barriers, which are still very effective based on the hierarchy of controls. We then reconsidered our barriers from activity #2, and came up with new barriers that were more effective. This activity taught us the different categories of barriers and the effectiveness they possess. With this knowledge, we once again applied it towards the near-miss analysis and also the bow tie method for hazard analysis because both require the development of barriers to reduce risks.

In activity #4, we were introduced to critical controls. A critical control is crucial to preventing an event or mitigating its consequences, its absence significantly increases the risks, and they prevent more than one type of unwanted event. We then went back to our PHA and identified which barriers were critical controls and classified them according to the hierarchy of controls. Our knowledge of critical controls learned in this activity was applied to developing our bow tie diagrams. Many barriers were proposed and to ensure we know which ones were the most important, we identified them as critical controls. These are explicitly mentioned in the bow tie section above.

Overall, this workshop strengthened our knowledge on safety and risk management, which allowed us to further develop our sensor to be safer for our users. It provided us crucial knowledge to include in other parts of our project that we would not have been able to do prior to this workshop. We also learned that eliminating risks is not always the goal, especially in science. As Lucca said in the workshop “risk is the price we pay to attempt to do great things.” So instead of our end goal to be risk free, we want to be more risk competent. This workshop was also a chance to educate all of our members on safety, and learn the importance of it.

The outline of the workshop is attached below so other iGEM teams can use it as inspiration for future safety activities. The outline also provides all the reference materials we used throughout the workshop which other teams can view and learn about the different aspects of risk management.

BIOSAFETY

E. coli JM109(DE3) and DH5α

We worked with E. coli JM109(DE3) and DH5α, both Risk Group 1 organisms commonly used for cloning, plasmid propagation, and protein expression. While these strains are considered non-pathogenic, the presence of recombinant DNA (e.g., antibiotic resistance markers, reporter constructs) required diligent containment and oversight.

Prevention & Containment

• All work was performed under BSL-2 conditions at designated benches.
• PPE included lab coats, gloves, and safety goggles; gloves were replaced immediately if torn or contaminated.
• Strict aseptic technique was enforced to avoid aerosol formation or cross-contamination between strains.

Sterilization & Waste Disposal

• Surfaces and instruments were disinfected with 70% ethanol before and after work.
• All disposable materials (pipette tips, agar plates, culture tubes) and reusable glassware were autoclaved prior to disposal or reuse.
• Biohazard containers were labelled clearly, and contaminated glassware was disposed of in sharps bins following institutional guidelines.

Strain-Specific Risks & Mitigation

• JM109(DE3) carries the T7 RNA polymerase gene, making it useful for protein expression but also more prone to unintended induction events. Care was taken to minimize stress conditions that could activate T7-driven unwanted expression.
• DH5α, optimized for plasmid cloning, is highly competent and thus more susceptible to accidental transformation. To mitigate this, plasmid handling was carefully segregated from other bacterial work.

Documentation & Traceability

• Glycerol stocks were prepared and catalogued in a digital database to ensure reproducibility and accountability.
• All strain/plasmid combinations were logged with freezer locations to maintain compliance with biosafety oversight.

MS2 Bacteriophage

Our project involved MS2 bacteriophage, a single-stranded RNA phage that infects E. coli. MS2 is Risk Group 1 and cannot infect humans, but viral particles pose specific containment challenges.

Prevention & Containment

• All manipulations of MS2 cultures were conducted at designated benches or biosafety cabinets to prevent accidental release.
• Pipetting was performed slowly to avoid aerosolization, a key risk factor for accidental spread.
• MS2 work was logged and scheduled separately from bacterial manipulations to prevent cross-contamination.

Sterilization & Waste Disposal

• All phage-containing materials (media, glassware, plastics) were autoclaved before disposal to ensure full inactivation.
• MS2 waste was segregated and labelled distinctly to protect autoclaving staff and avoid confusion with bacterial-only waste.
• Surfaces and pipettes were disinfected with ethanol and bleach after each session.

Phage-Specific Risks & Mitigation

• Although non-pathogenic to humans, MS2 could unintentionally infect E. coli cultures if released. To mitigate this, phage-only workflows were separated from cloning workflows.
• Because MS2 RNA can persist temporarily in the environment, additional emphasis was placed on surface decontamination and bleach-based sterilization.

Emergency Preparedness

All team members were trained in spill response and emergency procedures. Eyewash stations, spill kits, and emergency showers were identified at the start of lab work and remained accessible at all times. All personnel knew the procedures for handling accidental spills, broken contaminated glassware, or unexpected exposure. Routine refresher sessions ensured compliance and preparedness.

BIOSECURITY

Intro:

Our project focuses on the virus avian influenza, specifically targeting the viral RNA genome. The aim is to develop a rapid, easy to use, field deployable sensor to help detect the presence of the virus.

Before going into any details, we'd like to clearly define biosecurity. As mentioned before every website, article, and protocol will have different definitions because there is no standard definition for any vocabulary in safety. We have decided to use the definition from the World Health Organization (WHO) because we feel it is best suited for our project: “Policies, principles, technologies and practices implemented for the protection and control of and accountability for biological material, technology and information or the equipment, methods, skills and data related to their handling. Biosecurity aims to prevent intentional or accidental unauthorized access to, and loss, theft, misuse, diversion or release or even weaponization of such commodities” [3]. This definition encompasses both intentional and accidental misuse, loss, theft, etc. We noticed many biosecurity definitions only focus on either the intentional release of biological agents, or they have a very general definition summarizing its release without explicitly stating whether it is intentional or not. This definition by the WHO outlines both scenarios, which we felt that both the intentional and accidental practices should be outlined, as both are important to mitigate.

We chose to target the H5N1 avian influenza strain because it is an emerging disease that has become more relevant, and begun to affect the agriculture industry. However, the egg industry is not the only field struggling. The virus has also mutated in cattle, creating risks for the dairy and beef industries (information shared by Dr. Mohammed Faizal Abdul Careem, HP meeting). Viral testing on farms can be carried out by third-party veterinary diagnostic companies, farmers themselves, or the Canadian government if an official investigation is underway (as explained by Jenna Griffin, HP meeting). Looking ahead, a potential application of the sensor would be to monitor water bodies and other environments for wildlife carrying H5N1, as a preventative measure against outbreaks. This is particularly important because H5N1 is highly contagious and has an estimated 90% mortality rate (noted by Dr. Mohammed Faizal Abdul Careem, HP meeting).

Beef

Avian influenza is considerably less of a concern in the beef industry because it has not affected beef production as much as it has dairy and poultry. It has been confirmed that H5N1 has mutated to cattle, however, so far, there have been no confirmed cases of H5N1 in beef cattle in Canada or the U.S [4]. The beef industry understands the risks of avian influenza, however nothing has been implemented to reduce these risks. A meeting with Sean Thompson and Lorna Baird from Olds College in Alberta was conducted to gain insight on how beef farms are addressing the challenges produced by avian influenza. It was learned through this meeting that beef farms have many standard biosecurity protocols for other viruses and bacteria, however none for avian influenza. Most farms are reactive and passive as opposed to being proactive.

The Rumino sensor can act as a valuable point-of-care (POC) tool for early detection in beef farms. It can be used by third party veterinary diagnostics for monitoring potential exposure of the virus in cattle populations. Instead of requiring continuous on-site monitoring infrastructure, it provides a practical and rapid testing option that can be deployed as needed by veterinarians or farm staff. Third-party veterinary diagnostics can use our sensor for quick screening of cattle populations to monitor potential exposure to the virus. By integrating this sensor into current biosecurity protocols and also new ones for H5N1, beef farms can shift from purely passive surveillance toward proactive, event-driven testing. While this sensor currently functions as a POC tool, insights gained from its deployment will guide the development of more continuous monitoring systems in the future. This staged approach helps reduce the likelihood of outbreaks, enhances preparedness, and lowers the risk of cross-species infection.

Dairy

After a meeting with Claire Bertens, it is certain that the H5N1 virus has mutated from birds to cattle. H5N1 has a more significant impact on dairy cows than on beef cattle, largely because of its effect on milk production. It also has a higher retention rate in cattle compared to poultry. During infection the cow's appetite and milk production declines. Even after complete recovery the milk production does not return to original levels and the cow does not gain any resistance to the virus. Milk is the primary route for virus shedding, however pasteurizing the milk kills the virus making it safe for consumption, therefore raw milk is the main concern. Any raw milk runoff or waste from the dairy cattle is typically deposited into a large lagoon. The lagoon becomes a site for viral accumulation of H5N1, which potentially contributes to environmental spread and broader transmission risk.

Currently the dairy industry has a highly pathogenic avian influenza (HPAI) survey that shows where an infection occurred. Since any reported infection results in mass viral testing, farmers object to reporting because it restricts progress and their livestock may be killed. This survey is not mandatory so farmers tend to not engage because it is a sensitive topic.

The Rumino sensor will enable rapid on-site detection of the H5N1 virus without needing expensive and timely laboratory based systems. By streamlining viral diagnostics and reducing turnaround time, this sensor enhances the ability of third party veterinary diagnostics to track outbreaks, assess presence of viruses in livestock populations. This will strengthen surveillance infrastructure and early outbreak detection without requiring extra reporting from the farmers. For future applications, farmers can have test kits on hand and test the raw milk and nearby water bodies or areas where wild birds congregate. This will increase the biosecurity on dairy farms without the need for reporting because no livestock was tested.

Poultry/Egg

The poultry and egg industry has arguably had the most challenges facing avian influenza. Partly because the livestock in this industry is where avian influenza originated from. This year we went to visit a farmer, Mike from Rosalind Colony, where he shared that if he were to use a virus sensor, he would not test the chickens because then it would need to be reported. However, nearby water bodies and feces from other bird species such as geese would be of interest to test so the farmer knows where to isolate the flock from.

In Mike's case, he finds this sensor to be a tool that he will use to test feces, and other areas of his farm. Once again almost all livestock testing is done by third party veterinary diagnostic companies, and in this case Mike would also find the sensor useful for his own use as well. Current testing requires a laboratory and the turnaround time is about 1-3 days [5]. The Rumino sensor would allow for faster results so in the case of a positive outcome biosecurity measures can be carried out efficiently [6].

For a specific example, there is a large water body near Rosalind Colony that attracts many wild geese. As mentioned before, all of these wild birds are assumed to be infected with H5N1, therefore it is assumed the water body is also infected whether it is true or not. However, they have a system to treat their water where at one point the water decreases to a pH of 2. Due to the acidity of this condition, the virus will not survive this system so testing drinking water is not the primary concern. The main concern would be that the nearby pond becomes a hidden reservoir for the virus. This is where an environmental monitoring system would benefit Mike. Mike was particularly interested in testing geese feces nearby or on his farmland. Mike also has biosecurity protocols set in place, however he needs a system to notify him that there are infected birds nearby.

Summary of how Rumino can help the Agriculture Industry as a whole:

Viral biosensors play a critical role in strengthening the biosecurity against emerging threats like H5N1 avian influenza. This context is important because biosecurity is not just about individual farms, but also the systems, technologies, and practices that keep animal health, food security, and even public health safeguarded. The WHO has warned that the next pandemic will be caused by influenza, and the Canadian Food Inspection Agency (CFIA) has identified that improved diagnostics and environmental testing is a priority for highly pathogenic avian influenza (HPAI). As a rapid POC tool, Rumino directly addresses these needs by offering fast, reliable results that integrate smoothly into veterinary diagnostic workflows while avoiding additional reporting burdens for farmers.

How Rumino contributes to Environmental and Community Risk Mitigation

Looking ahead, extending this sensor into environmental applications would be beneficial as it would provide farmers with proactive surveillance capabilities, early confirmation of areas of infection, and reinforcing existing biosecurity protocols. In this way, everything we have outlined (from early detection to integration into biosecurity frameworks) shows how Rumino supports a shift toward preventative, system-wide protection against influenza outbreaks. Although this sensor is currently specified for H5N1 avian influenza, it can be easily altered to detect other viruses by changing the substrate and incumbent oligonucleotide strands.

Dual-use Concerns (bacteriophage)

Our project is not just about developing a biosensor, but also about creating a phagemid-based proof-of-concept system. A biosecurity risk for our phagemid is the potential for dual-use. The phagemid is intended to produce ssRNA so we can validate our system without using pathogenic viruses or more expensive diagnostic solutions. However, it has the potential to be misused to produce viral RNA genomes for malicious activities. Although the phagemid is already well characterized and used among many biologists, we are modifying it to make it more modular. The modularity comes from allowing the ssRNA being produced to be more customizable so it is easier to use. This is also where the dual-use concern arises. Although for our purposes, we are using it to create synthetic oligonucleotides for validation of our sensor, if misused it can be used as a bioweapon to create viral RNA sequences that resemble pathogenic viruses to infect humans. Acknowledging these risks highlights why we have outlined our detection approach, environmental testing rationale, stakeholder feedback, and phagemid proof-of-concept: together, they show how the Rumino sensor can be realistically deployed as a point-of-care system validated with safe, modular ssRNA controls. This integration ensures the device can fit into veterinary workflows while aligning with broader biosecurity priorities.

A meeting with Research Security highlighted the importance of discussing dual-use potential of technology. In the beginning, we were considering not mentioning dual-use as a whole because the potential misuse of this sensor or the phagemid seemed very unlikely. Research Security experts Cynthia Omajugho and Matthew Gale both emphasized that even the most unlikely dual use concerns must be discussed because many of the consequences end up causing serious harm physically, mentally, or socially.

Mitigating Risks:

Human Infection: Bacteriophages were long believed to infect only bacteria. More recent studies suggest that some phages may cross epithelial barriers via endocytosis and reach internal organs [7]. For MS2 specifically, recognition by toll-like receptors (TLR7/8) has been described, although this remains hypothetical and has not been demonstrated in real-world cases [7].

Risk Mitigation:

• All work with MS2 was conducted under standard laboratory biosafety procedures. Autoclaving and bleach disinfection reliably destroy phage particles [8].
• The plasmid design excludes antibiotic resistance markers, preventing survival or selection outside controlled conditions, in line with iGEM Safety and Security Recommendations [9].

Replicating viral genomes: MS2 has a very limited genome capacity, able to package only ~3 kb of ssRNA [10]. In comparison, medically important viruses such as avian influenza, measles, Zika, and HIV-1 all require much larger genomes [11–14].

Risk Mitigation:

• When choosing the MS2 phage, we considered the strict 3 kb packaging limit ensures MS2 cannot replicate a full viral genome. Any packaged RNA fragments would lack essential coding regions and be non-infectious [10].
• We also took into account MS2 replication also depends on its own highly specific replicase, which is incompatible with heterologous viral genomes, preventing unintended amplification [15].

Unintended Dissemination: MS2 was chosen because it is non-enveloped, giving it greater resistance to some chemical and physical stresses [16]. Despite this stability, it is still sensitive to heat, with capsid breakdown occurring at elevated temperatures [17].

Risk Mitigation:

• All work with MS2 was conducted under standard laboratory biosafety procedures. Autoclaving and bleach disinfection reliably destroy phage particles [8].
• We took into account that, while the capsid stabilizes the genome, once disrupted, the RNA is extremely fragile and rapidly degraded by RNases in the environment [18].
• Lastly, we made the choice to use the MS2 phage as it is widely used as a safe surrogate in environmental virology, confirming its lack of pathogenicity to humans [19].

While the risk of malicious use of the phagemid is low, it is not absent. By engaging with Research Security at the University of Calgary, even extremely low probabilities of dual-use potentials should be discussed due to the harm it could potentially cause. These harms are usually very serious and can cause distress and fear or be a potential weapon used against the community. We acknowledge the dual-use concerns such as synthesis of viral RNA and potential of human infection to embed strict controls (e.g. MS2 genome packaging limits, controlled strain access, and exclusion of antibiotic resistant markers) to prevent misuse of the phagemid. We have addressed both, intentional (e.g. viral RNA synthesis), and unintentional (e.g. unintended dissemination) misuse pathways to ensure optimal risk management for dual-use.

HAZARD ANALYSIS

With the guidance from Lucca C. Filippo, the bow tie method was selected for our hazard analysis because it provides a clear, visual, and structured way to map the relationships between hazards, threats, consequences, and barriers in our system [20]. Unlike text-heavy safety documentation, the bowtie diagram condenses complex risk scenarios into a format that is quick to interpret, making it more accessible to both technical and non-technical audiences [20]. The bow tie method focuses on the operational aspects of a system and highlights the barriers to reduce the risks [11]. This approach is particularly valuable for our project, as the phagemid-based components and Rumino sensor require careful operational risk management [26,27,29].

Figure 2. Visual diagram/legend of our bowtie analysis

When developing the bow tie, there are several different components to the bow tie diagram. All of which are important to understand before creating and interpreting the diagram. The parts of the bow tie are all essential since the goal of a bow tie is to highlight the relationships between them. The components of the bow tie include:

• Hazard: An operation, activity or material with the potential to cause harm to people, property, environment or business; or simply, a potential source of harm [21]. This will be the definition of a hazard used throughout the entire safety section. A hazard can be as simple as ‘driving a car’ because operating a vehicle is a hazard itself even though it is very common.
• Top Event: The moment when control of the hazard is lost; there is no damage done yet, but it is imminent [20]. Using ‘driving a car’ as the hazard for an example, the top event can be ‘losing control over the car’ [20].
• Threats: Potential reasons for loss of control of the hazard leading to the top event [20]. A threat is anything that could cause the top event. Continuing with the car example, a threat could be ‘intoxicated driving’.
• Consequences: A result of the top event. These are the possible outcomes that can occur once the top event happens [20]. An example for the top event “losing control over car”, a consequence is ‘car crashes into a light post’.
• Barriers: Barriers are the controls of these unwanted scenarios. There are two types of barriers: prevention and mitigation.
  • Prevention: Controls to prevent the threats from occurring [20]. These barriers must be able to 100% prevent the threat from occurring, but do not necessarily need to be 100% reliable [21].
  • Mitigation: These are employed after the top event occurs to either prevent the consequence from occurring or reduce the losses of the consequence [20, 21].
• Escalation/Degradation Factors: A condition that leads to impairment, failure or reduces the effectiveness of the barrier to which it is attached [21]. In simple terms, an escalation factor is anything that can cause the barrier to fail. Escalation factors do not cause threats or consequences; they instead degrade the main barrier.
• Escalation/Degradation Control: An escalation control prevents the escalation factor from occurring [21]. It is simply a barrier for an escalation factor.

Part of the bow tie method is to analyze relationships between the different components. Many of the bow ties we create will have components that move forward into other bow tie diagrams as well. For example, the consequence “glass breakage” for one bow tie will also be a top event for another. This shows the connection of hazards, top events, consequences, etc associated with our system.

The hazards and risks for this sensor will inevitably change because the product is expected to be further developed and go through many iterations. As the system changes, the bow ties will as well. This considered, there should be a hazard analysis for the beginning of a project, and it will be updated as the product evolves.

Our team chose to perform a bow tie analysis instead of continuing the System Theoretic Process Analysis (STPA) because we want to depict the relationships between hazards, threats and consequences in a visual way. Over the course of our iGEM journey, we learned that many individuals find safety to be a burden and almost like an ‘obstacle’ for them to do research, despite the importance of safety. So we decided to go for a more visual approach that is eye pleasing, easy to understand, but still contains all the necessary information.

The objectives of our analysis were to:

• Identify common/recurring hazards associated with the handling, storage, packaging, and disposal of phagemids and the Rumino sensor.
• Map the threats, consequences, and barriers for each hazard in a visual format to make their relationships clear and accessible.
• Determine the most critical barriers (e.g., biosafety cabinets, validated inactivation protocols, colour-coded labelling systems, and spill response kits) and highlight their role in reducing systemic risks across multiple hazards.
• Highlight procedural gaps and develop targeted improvement strategies that align with institutional, national, and iGEM best-practice biosafety standards.
• Develop a safety plan that directly addresses identified gaps through short-term, medium-term, and long-term actions.

Methodology

When developing each diagram, the methodology from the book Bow Ties In Risk Management: A Concept Book for Process Safety was used to ensure consistency and repeatability of the bow tie method. Following a systematic method also allows for completeness, effectiveness, and efficient communication.

The hazard identification (HAZID) process began with a systematic review of all laboratory activities involving phagemids, user activities with the sensor [2,28,26,27,29]. This is the first step to any bow tie method.

Connecting to the hazard, each bowtie diagram was constructed with a central top event representing the moment when control over the hazard is lost [20]. The left side of the diagram lists threats with corresponding preventative barriers, and the right side shows potential consequences of the top event, with corresponding recovery barriers [20,24,25]. Escalation factors were noted for any barrier that could be weakened by a certain event, along with escalation controls to prevent these weaknesses from developing [20,24,25].

During the “Risk Management Workshop” with graduate student Lucca C. Filippo, we went through the different steps of a preliminary hazard analysis. We were introduced to formal safety terminology and trained in structured hazard identification methods. This includes outlining hazards, consequences, barriers, as well as likelihood/severity. Although this workshop was not specific to the bow tie method, the key ideas of risk management still highlighted key components of the bow tie, which helped in the development of the diagrams. This workshop helped us differentiate differences between hazards and consequences, and implement different barriers to mitigate risks.

The workshop also introduced the idea of critical controls, which are essential to mitigating risks of a specific hazard. These barriers are the most recommended or absolutely necessary to ensure safety of people, environment, and production. Critical controls of each hazard were identified using the following criteria:

1. It is crucial to preventing an event or mitigating its consequences.
2. Absence or failure significantly increases the risks, despite there being other controls.
3. Prevents more than one type of unwanted event.

Rumino Sensor Bow ties

The Rumino utilizes biomolecules and chemicals to detect a specific analyte usually at very low concentrations [22]. In our case, it uses a glass capillary tube lined with ssDNA that is modified with hydrophobic groups to detect the presence of other oligonucleotides (i.e., RNA/DNA genomes). The use of biomolecules and chemicals are a hazard for this project, especially because this is a hand-held manual device. A bow tie was created to analyze the potential consequences if a user were to be exposed to these molecules in the system.

Figure 3. Bow tie diagram for hazard “Chemical and biological agents in the sensor”

Samples being tested are all potential carriers of the H5N1 virus, making each sample a potential hazard despite its uncertainty. The reason we are treating every sample as a hazard is so that we can ensure optimal safety of the user. In addition, the CFIA recognizes wild birds as a natural reservoir for avian influenza, and that water sources contaminated by these birds can act as a medium for transmission [53]. In our case, it uses ssDNA to detect the H5N1 virus based on the presence of its ssRNA genome. The main point of concern is during sample collection using the cotton swab because the raw sample is what contains the active virus. The sample preparation buffer contains lyophilized proteinase K which lyses the virus, essentially neutralizing it, eliminating this potential threat.

Figure 4. Bow tie diagram for hazard “Testing a potentially virulent sample”

This hazard is often overlooked. Despite the common use of glass items, it still carries risks that are often overlooked due to its daily routine use. Frequent use of glassware fosters an intuitive understanding of glass handling practices such as not holding it too tightly, ensuring not to drop it, and placing it down gently. All of these are controls/barriers that we have developed; however, they will still be outlined for completeness of the analysis, but also as friendly reminders. Besides cuts and scratches, in our case, glass breakage can also result in contamination of biological and chemical molecules, making it an important hazard to analyze.

Figure 5. Bow tie diagram for hazard “Operating a glass device”

Chemicals will often affect both the environment and humans. Some chemicals will only affect the environment, while others only humans; however, many affect both as well. The risks of the chemicals towards the environment have already been outlined in a previous bow tie, so this one primarily outlines the risks of physical human health. The controls/barriers for this bow tie are primarily PPE. Although PPE is the least effective type of control according to the hierarchy of controls, it acts as a physical barrier even though it does not remove the risk itself.

Figure 6. Bow tie diagram for hazard “Chemicals in capillary tube or buffer”

Phagemid Bow ties

Cloning bacteria is a very common and well-developed laboratory technique in synthetic biology, so its risks are well controlled, but not eliminated. It is unrealistic to completely eliminate risks in the laboratory since it would be impossible to study biology without them. However, we can try to reduce the consequences of their losses by including more barriers. The specific top event we are analyzing from this hazard is the escape of the phagemid during its production. This is an essential risk to analyze, as accidental release of the phagemid would result in more serious consequences, outlined in the bow tie.

Figure 7. Bow tie diagram for hazard “cloning bacteria containing phagemid”

Transport and packaging of biological organisms presents many risks ranging from accidental release due to packaging errors to degradation of biological agents during storage, potentially compromising the integrity and usability of the product. Although this is not a hazard directed to other iGEM teams working in the lab, it is a potential hazard during its production. When doing a bow tie analysis, it is also important to consider the impact of hazards and consequences outside of safety and biology. This would be especially useful from an entrepreneurial side because it must be considered when the phagemid is sold and shipped to the consumer.

Figure 8. Bow tie diagram for hazard “phagemid packaging and handling”

After the phagemid is expressed, it possesses the ability to infect bacteria and produce ssRNA. Handling an engineering product will have its own risks because it is capable of its own activity. Differences between this bow tie analysis and the one for “cloning bacteria containing phagemid” are mainly the exposure to humans compared to the environment. This bow tie is aimed to analyze the consequences of human exposure to the phagemid.

Figure 9. Bow tie diagram for hazard “Handling engineering material”

Once again, the above bow ties were evaluated independently, but several themes still emerged when reviewing them collectively. We have summarized patterns across hazards, highlighting recurring threats, systemic weaknesses, and shared strengths in our biosafety framework. By identifying these overlaps, we can prioritize actions that address multiple risks at once, maximize the impact of our safety measures, and strengthen the overall resilience of our phagemid workflow.

Rumino Sensor

• Recurring Threats:
  There were several threats appearing across different bow ties analyzing the Rumino sensor. These include contact with chemicals, from handling the sample buffer or using the capillary tube. Glass breakage was also a common threat because it can result in exposure and leakage of chemicals and potential minor first aid injuries, such as minor cuts and scrapes. There are a few hazardous chemicals used in the sample preparation buffer, as well as modifying glass surfaces of the capillary that makes these threats more serious.
• Systemic Weaknesses:
  Many threats can be resolved by following protocols properly. A small pamphlet is included in the product to guide the user on how to use the sensor properly, accurately, and safely to mitigate this concern. However, we are unable to oversee how the product is being used once it is beyond our facility. This creates a systemic weakness because an unlimited amount of protocols can be made and given, however there is no guarantee that they will be followed unless there is constant supervision.
• Strong Barriers:
  Developing barriers that were all effective, independent, and auditable was a challenge. However, it was found that once a barrier met all three criteria, they made for very strong barriers even if they are seemingly simple. There are many barriers that appear in multiple bow ties such as proper use of PPE, proper hygiene, and following proper protocols. Despite these barriers being seemingly simple, without them the risks of using the Rumino sensor would increase because something as simple as a glove could prevent chemical burns, allergic reactions, or skin irritation due to chemical exposure.

Phagemid

• Recurring Threats:
  Several threats were identified as appearing across multiple hazards, indicating systemic vulnerabilities in our phagemid work. Phagemid Packaging and Handling, and Containment Loss from Cloning Bacteria, spillage or aerosolization during pipetting or centrifugation also recur in both Cloning Bacteria Containment and Phagemid Packaging, highlighting the need for consistent aerosol-control practices. Additionally, concurrent work with high-risk organisms and cross-contamination between strains are repeated threats that span operational and biological hazard categories.
• Systemic Weaknesses:
  Across hazards, common weaknesses include gaps in staff training (especially refresher and competency assessments), incomplete or inconsistent documentation, and insufficient enforcement of SOPs. Several hazards cite inadequate recordkeeping or rapid team turnover as contributing factors to regulatory noncompliance and loss of research reproducibility. The lack of standardized SOP templates and centralized tracking systems is evident in both Documentation & Compliance and operational hazards, indicating a need for system-wide process standardization.
• Strong Barriers:
  Some barriers are consistently effective across multiple hazards. Routine biosafety training records and competency checks address threats in both personnel safety and operational hazards. Strict waste decontamination protocols (autoclaving, bleach) and biosafety cabinet use for aerosol-generating steps serve as strong physical and procedural controls for containment-related hazards. Material transfer agreements also appear in both personnel safety and documentation hazards, helping prevent misuse or unregulated sharing of engineered materials.

Our Safety Implementation so far

While each hazard in our bow tie analysis was evaluated independently, several themes emerged when reviewing them collectively. This section summarizes patterns that cut across multiple hazards, highlighting recurring threats, systemic weaknesses, and shared strengths in our biosafety framework. By identifying these overlaps, we can prioritize actions that address multiple risks at once, maximize the impact of our safety measures, and strengthen the overall resilience of our phagemid and sensor workflow.

Rumino Sensor

Creating and Including Protocols

• A thorough pamphlet outlining the safety precautions, waste disposal instructions, handling precautions, and ingredient list has been created and will be provided upon purchase of the test kit.
• Waste protocol is designed to prevent harm to both humans and the environment.
• Protocols are created in accordance with Canadian environmental regulations, specifically addressing phosphates and hazardous chemicals.

False Result Mediation

• A negative control tube will be provided in the test kit to act as an indicator of what a sample should look like without the presence of the H5N1 avian influenza strain.
• Visuals are included in the pamphlet to show what positive, negative, and control samples should look like upon testing, ensuring users know what to look for rather than relying solely on written descriptions.

Potential Infection

• Sample buffer contains chemicals that lyse the virus and neutralize it so it no longer has infectious capability.
• The use of gloves is clearly outlined in protocols to ensure no direct contact with the sample occurs prior to treating it with the sample buffer.
• Plastic bags are provided in the test kit to ensure there is a physical barrier between used materials and the user.

Phagemid

Containment and Waste Management

• All engineered material is inactivated through strict waste decontamination protocols (autoclave, bleach) before disposal.
• Dedicated pipettes and filter tips are used for each strain to prevent cross-contamination.
• Biosafety cabinets are required for all aerosol-generating procedures.
• Clearly labelled containers, tube color coding, and hazard signage minimize handling errors and accidental misuse.
• Environmental sampling and immediate response protocols allow early detection and containment of accidental releases.

Personnel Protection and Training

• Mandatory use of gloves, lab coats, and eye protection protects staff from direct exposure.
• PPE spot-checks, donning/doffing training, and a no open skin policy reinforce proper protective equipment use.
• Staff receive initial biosafety training, with competency assessments and regular refresher modules on GMO handling and Good Microbial Practices (GMP).
• Training records are maintained to verify that all members meet baseline biosafety standards.
• Immediate cleanup and reporting protocols are enforced for spills, supported by readily available spill kits.

Documentation and Oversight

• Standardized SOPs are in place for phagemid preparation, instrument decontamination, and session checklists.
• Quarterly SOP reviews and visibly posted instructions keep procedures current and accessible.
• A centralized digital record system with backups preserves safety documentation and experimental records.
• Plasmid maps, safety sheets, and version-controlled records are maintained in Benchling to improve traceability.
• Risk assessments are guided by a standardized template and submitted for review by the Institutional Biosafety Committee before work begins.
• Internal compliance audits are conducted annually to identify and correct potential gaps.

Biocontainment Safeguards

• Immediate inactivation protocols ensure phage particles are neutralized after use.
• Use of auxotrophic strains and biocontainment systems restricts engineered bacteria from surviving outside the laboratory.
• Work with phagemids is separated from phage-free zones using dedicated equipment to prevent cross-contamination.

RumiSafe

As part of our commitment to advancing safety in synthetic biology, our team developed a user-friendly bowtie analysis tool. This software allows teams to quickly and clearly map out hazards, threats, consequences, and barriers using the internationally recognized bowtie risk assessment framework.

The motivation for this contribution came from our own experience: when working through safety and security forms, we found that most available risk assessment tools were either too complex, locked behind paywalls, or requiring an account. We wanted to create something accessible, intuitive, and freely available for all iGEM teams. Additionally, when making our bow tie diagrams, we found it to be easy, however very time consuming and inefficient due to the constant editing of lines and boxes. This tool will allow other teams to make bow tie diagrams with ease without losing any of its detail.

Our software provides:

• A simple interface where users can build bowtie diagrams step-by-step.
• Built-in categories relevant to iGEM projects (biological, operational, environmental, ethical, etc.).
• Export options that allow teams to directly include their diagrams in wiki pages, safety forms, and presentations.
• A framework for capturing not only hazards and consequences, but also barrier strength, escalation factors, and recovery measures, making analyses both detailed and auditable.

By making safety analysis more approachable, our contribution lowers the barrier for other iGEM teams to engage deeply with risk management and supports the iGEM Safety & Security Committee’s goal of fostering a culture of safety across the community.

How Other Teams Can Use It

1. Access the tool: https://gitlab.igem.org/2025/software-tools/ucalgary
2. Define your hazard and top event.
3. Add threats and consequences.
4. Insert preventative and/or recovery barriers.
5. Add escalation factors.
6. Insert escalation factor barriers.

We also provide example files based on our own project (as seen above), which can serve as a template for other teams to learn from.

Technical Development

• The software's architecture is made up using Python and Dash by Plotly.
• A key design choice was to make the software very user-friendly and for users of all experiences. In order to achieve this, we utilized a sidebar that allowed the user to access all core functions.
• The main challenge that was faced was the alignment and layout of the software. It was integral that the software was visually intuitive for the user, so ensuring that the visual portion of the software was responsive was the top priority. This was solved using responsive values in CSS to ensure that the alignment stayed consistent across various screen sizes.
• In the future, we hope to include more consequences and threats since we limited them to 10. We would also love to add an export feature that allows the user to export the bowtie they have created in various formats.

Looking Forward

We hope this software will become a long-lasting resource for the iGEM community. By lowering the barrier to perform structured safety analysis, we aim to not only make iGEM projects safer, but also empower teams to think critically about risks, responsibilities, and resilience in synthetic biology.

PRODUCT SAFETY

Safe by Design/Iterations

Iteration 1: In-vivo detection using mesoporous nanoparticles (MSNPs)

In the very beginning of our iGEM journey, we wanted to work on in-vivo detection for humans using MSNPs, however we quickly moved away from this mainly due to the feasibility, but there was also a safety concern that was realized shortly after. Nanoparticles (NPs) have been of interest to scientists for many years for the purpose of drug delivery. Despite their popularity, their safety towards humans has still been questioned due to their potential toxicity to organs. Since using MSNPs were only considered for a short amount of time, the size and retention time in the body of the nanoparticle was not determined. Due to this ambiguity, the safety of our first iteration is difficult to touch on because if a small 10nm MSNP was used there could be serious safety concerns because smaller NPs can easily be taken up by cells, which leads to cytotoxicity [30].

Iteration 2: Gold Nanoparticle Lateral Flow Assay (LFA)

We chose very early on that our goal for the end of the summer was to develop an operational product. To ensure we had a feasible project and a viable product we switched to develop an in-vitro biosensor in the form of a lateral flow assay that resembles a rapid COVID-19 test. Switching to in-vitro detection eliminates many of the health concerns in-vivo detection may have brought. For testing outside of the body, there is no synthetic material going inside a human, nor is any lab work or extensive machinery required for the end product. With this version most of the risks can be mitigated through the use of PPE or proper hygiene.

Iteration 3: Manual Capillary tubes based Sensor

The jump from a LFA to making a capillary based system was not due to safety purposes, but more so for novelty. In the beginning we decided to use hydrophobic groups to change the wettability of the glass surface to cause a height change in solution. However experimentation to modify the DNA with hydrophobic groups was not very successful. This considered a new version was proposed using methylene blue to release a colorimetric signal. This was quickly discontinued as methylene blue is a toxic dye, making it a hazard for users and the environment in high concentrations [31,32]. The only safe way to consume methylene blue is to treat methemoglobinemia, any other way can cause health issues such as serotonin syndrome when contaminated with other serotonergic drugs and can also central nervous system symptoms such as dizziness and confusion [31]. Methylene blue is also not biodegradable, so it can persist in the environment. This would cause damage to marine life as the dye would prevent sunlight penetration in water bodies [32].

This considered, the idea on methylene blue was discontinued and development using hydrophobic groups was continued. Without methylene blue the components of this capillary based biosensor are much safer to use.

Test Kit Components

The test kit for the Rumino sensor is very minimal and simplistic so it is easy to keep track of and use. It consists of the capillary, a buffer, and different consumables and containers to ensure safe and easy use of the sensor. Before proceeding to a detailed explanation of each component, it is important to keep in mind that every chemical and biological material carries some level of risk, even if they are relatively safe. Nothing in biology, nor science in general, is completely free of risks, so it is impossible to create a product that is 100% safe. However, the chemicals in our kit were chosen to minimize the potential hazards associated with their intended use. For the chemicals that are hazardous, there will be clear guidelines included in the kit to outline safe handling. This allows the user to handle our product safely without sacrificing the effectiveness.

When discussing the different chemicals and biological molecules, their safety data sheets (SDS) will be referred to. SDSs by Sigma Aldrich and IDT will be the ones referred to as they were found to be the most updated and most of the chemicals and DNA in our lab were purchased from them. Both companies follow the OSHA Hazard Communication Standard (HCS) when classifying hazards of products and also their safety. According to the OSHA HCS, chemicals are hazardous if they present any physical or health hazard:

• Physical Hazard: “a chemical for which there is scientifically valid evidence that it is a combustible liquid, a compressed gas, explosive, flammable, an organic peroxide, an oxidizer, pyrophoric, unstable (reactive) or water-reactive.” [33]
• Health Hazard: “a chemical for which there is statistically significant evidence based on at least one study conducted in accordance with established scientific principles that acute or chronic health effects may occur in exposed employees. The term 'health hazard' includes chemicals which are carcinogens, toxic or highly toxic agents, reproductive toxins, irritants, corrosives, sensitizers, hepatotoxins, nephrotoxins, neurotoxins, agents which act on the hematopoietic system, and agents which damage the lungs, skin, eyes, or mucous membranes.” [33]

Capillary tube components:

Table 3. All components making up the capillary tube based sensor and a description about how and why it is safe, and the risks it possesses.

Component	Safety Description
Lyophilized DNA	The substrate strand is complementary to the viral genome (target) so it can undergo the toehold mediated strand displacement reaction. DNA itself is not a hazardous substance so it will not cause physical or health related harm to people [50]. The risk comes from the potential for other organisms to take up this DNA once it leaches into the environment. This could cause unintentional gene transfer between other wildtype organisms potentially resulting in unpredictable mutations.
3-Aminopropyltriethoxysilane (APTES) modified glass capillary tube	This is the main compartment where the detection of the virus occurs. The capillary tube holds the solution and the glass surface is functionalized with APTES in order to immobilize the DNA. The APTES will be bound to the inside of capillary tubes, so there is no necessary contact with the APTES. The main concern are free APTES molecules which will only be present if hydrolysis of the bound APTES occurs. This is unlikely because this reaction requires exposure to water molecules for longer than the test requires.
Dodecanol/Dodecane-amine	Used as the hydrophobic group to change the wettability of the glass surface to cause a visible height change in the sample solution. Dodecanol can cause serious eye irritation, however the LD50 is >5000mg/kg for oral consumption and >8000mg/kg for dermal, which according to the Canadian Centre for Occupational Health and Safety (CCOHS) is practically non toxic to humans, making it relatively safe to handle [51]. However, dodecanol is very toxic to aquatic life so proper disposal of this is required to protect the environment [51].
Glutaraldehyde	Used as a linker to attach the APTES modified glass to the amine modified DNA. Free glutaraldehyde is a very toxic substance, however similarly to APTES, it is covalently bound to the inside of the capillary, so there is no necessary contact with the molecule.

Buffer elements:

All of these are further analyzed with a bow tie. *All elements (except ethanol) are lyophilized to stabilize the chemicals and proteins to allow for longer storage.

Table 4. All components making up the sample preparation buffer and a description about how and why it is safe, and the risks it possesses.

Component	Safety Description
Disodium Hydrogen Phosphate + Potassium Hydrogen Phosphate	Used to make a PBE buffer for pH regulation so the solution is stable. This chemical is considered non hazardous so no physical or health hazards are present for humans [35]. The main concern for phosphate buffers is not its toxicity, but environmental contamination. Excess phosphates in water bodies promote algal growth, resulting in eutrophication, causing damage to marine life [36].
Sodium Chloride + Potassium Chloride	Both NaCl and KCl are classified as non-hazardous chemicals [37,38]. They pose no serious effects on human health making them very safe for us to handle.
75% Ethanol	Ethanol was carefully chosen opposed to sodium hypochlorite (i.e. bleach) as a safer alternative. Bleach may be more effective at viral lysis, however it is a corrosive and toxic chemical to humans and aquatic life, and can cause chemical burns and irritation when it comes into contact with skin [40]. Although ethanol is flammable and can also cause eye irritation, it is much safer in respect to bleach [39].
Proteinase K	Proteinase K is a common enzyme that lyses viruses. For our purposes we need to lyse the avian influenza virus to obtain its RNA genome. By lysing the virus, it allows us to extract the analyte for our sensor (RNA) and it simultaneously also kills the virus by destroying the capsid and other outer protective layers. Proteinase K is a health hazard that causes serious respiratory sensitization [44]. In a lyophilized state, it can survive in pH ranges from 4–12 and temperatures up to 65°C [45]. Safe handling of this protein is required to ensure safety of all users.
N-acetylcysteine (NAC)	NAC reduces viscosity of sputum so it can more easily dissolve in the buffer. Originally we wanted to use dithiothreitol (DTT) as it can reduce sputum viscosity, lyse the virus, and increase proteinase K activity. We decided to substitute it for NAC because NAC is a safer substitute [48,49]. While DTT is effective, it is a corrosive chemical that causes serious skin, eye, and respiratory irritation with negative impacts to aquatic life as well [46,47]. Despite causing serious eye irritation, NAC is much safer since it is not corrosive, and has an oral LD50 of 5050mg/kg, which is categorized as practically non-toxic according to CCOHS [34,48,49].
Sodium Dodecyl Sulfate (SDS)	SDS is capable of viral lysis and nuclease inhibition, preventing the substrate, incumbent, and target strands that mediate TMSDR from being degraded. SDS is a slightly toxic, corrosive chemical capable of causing skin and eye irritation [52]. It also has flammable properties and is harmful when swallowed and inhaled [52].
Ca2+ (Calcium ions)	Calcium ions stabilize proteinase K. We will be obtaining calcium ions using calcium acetate. This is a water soluble and non-hazardous calcium source, making it a safer alternative to other calcium compounds such as CaCl [43]. CaCl is not inherently toxic to humans, however it is significantly more toxic than calcium acetate due to its hygroscopic nature [41]. Its ability to attract and absorb moisture causes serious irritation and even chemical burns [41,42].

The goal of the buffer is primarily sample preparation so the RNA genome can be extracted from the virus. However, the components were carefully chosen to ensure safety of the user. As mentioned before every chemical has its own risks, in our case most of the chemicals can cause skin and eye irritation to a certain degree, however mitigation strategies are outlined in the bow tie analysis as well as the warning label below. At the same time as prepping the sample for testing, this buffer lyses the virus, which essentially “kills” the virus, diminishes its ability to infect others. RNA is notoriously one of the most fragile compounds, so it will not be able to survive for long outside of the virus considering its fragile nature and the RNase enzymes present in the environment and on humans. This decreases the risk of horizontal gene transfer in the environment due to DNA/RNA contamination.

There are a few notable chemicals used to produce the sensor that are inherently toxic and some may be dangerous to handle if they become unbound from the glass surface. Most chemicals are safe for human handling, however many chemicals pose negative impacts to the environment. We feel that safety not only encompasses the user, but also their surrounding environment in which we all live in. This considered a waste protocol is provided in the test kit and it is expected to be followed strictly among users. The waste protocol was designed to ensure the safety of users, and the environment. Since we are mainly targeting the veterinary market, the waste disposal and handling procedure is tailored to their needs and also the facilities they have access to.

Each kit will include a warning label either stickered onto every kit produced or included as a mini pamphlet in the kit so users are aware of its hazards, and what to do in case of direct contact, accidental release, spillage, etc. This label includes instructions on how to use the sensor, as well as proper disposal protocols, which are both expected to be strictly followed. The protocol was made following Alberta and Canadian regulations and standards, making this widely applicable for any field product. This warning label was created based on the hazard analysis conducted using the bow tie method to ensure all hazards and risks are communicated to users.

REFERENCES

[1] International Organization for Standardization. ISO 35001:2019 – Biorisk management for laboratories and other related organisations. Geneva: ISO; 2019.

[2] World Health Organization. Laboratory Biosafety Manual. World Health Organization; 2020.

[3] World Health Organization. Laboratory biosecurity guidance. World Health Organization; 2024.

[4] American Veterinary Medical Association. Avian influenza virus type A (H5N1) in U.S. dairy cattle | American Veterinary Medical Association [Internet]. www.avma.org. 2025. Available from: https://www.avma.org/resources-tools/animal-health-and-welfare/animal-health/avian-influenza/avian-influenza-virus-type-h5n1-us-dairy-cattle

[5] Cleveland Clinic. PCR Test [Internet]. Cleveland Clinic. Cleveland Clinic; 2025. Available from: https://my.clevelandclinic.org/health/diagnostics/21462-covid-19-and-pcr-testing

[6] Canadian Food Inspection Agency. Highly pathogenic avian influenza (HPAI) science and research priorities - inspection.canada.ca [Internet]. Canada.ca. 2024. Available from: https://inspection.canada.ca/en/animal-health/terrestrial-animals/diseases/reportable/avian-influenza/latest-bird-flu-situation/hpai-science-and-research-priorities

[7] Podlacha M, Grabowski Ł, Kosznik-Kawśnicka K, Zdrojewska K, Stasiłojć M, Węgrzyn G, et al. Interactions of Bacteriophages with Animal and Human Organisms—Safety Issues in the Light of Phage Therapy. International Journal of Molecular Sciences [Internet]. 2021 Jan 1;22(16):8937. Available from: https://www.mdpi.com/1422-0067/22/16/8937/htm

[8] Guglielmotti DM, Mercanti DJ, Reinheimer JA, Quiberoni ADL. Review: efficiency of physical and chemical treatments on the inactivation of dairy bacteriophages. Frontiers in Microbiology [Internet]. 2011;2:282. Available from: https://pubmed.ncbi.nlm.nih.gov/22275912/

[9] Igem.org. 2025. Available from: https://responsibility.igem.org/guidance/working-safely

[10] Mikel P, Vasickova P, Kralik P. Methods for Preparation of MS2 Phage-Like Particles and Their Utilization as Process Control Viruses in RT-PCR and qRT-PCR Detection of RNA Viruses From Food Matrices and Clinical Specimens. Food and Environmental Virology. 2015 Feb 25;7(2):96–111.

[11] Sangsiriwut K, Uiprasertkul M, Payungporn S, Auewarakul P, Ungchusak K, Chantratita W, et al. Complete Genomic Sequences of Highly Pathogenic H5N1 Avian Influenza Viruses Obtained Directly from Human Autopsy Specimens. Stewart FJ, editor. Microbiology Resource Announcements. 2018 Dec 6;7(22).

[12] Bankamp B, Liu C, Rivailler P, Bera J, Shrivastava S, Kirkness EF, et al. Wild-Type Measles Viruses with Non-Standard Genome Lengths. Hahm B, editor. PLoS ONE. 2014 Apr 18;9(4):e95470.

[13] Nitatpattana N, Chaiyo K, Rajakam S, Poolam K, Chansiprasert K, Pesirikan N, et al. Complete Genome Sequence of a Zika Virus Strain Isolated from the Serum of an Infected Patient in Thailand in 2006. Genome Announcements [Internet]. 2018 Mar 8 [cited 2020 Nov 10];6(10). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5843728/#:~:text=The%20ZIKV%20genome%20is%20a

[14] van Heuvel Y, Schatz S, Rosengarten JF, Stitz J. Infectious RNA: Human Immunodeficiency Virus (HIV) Biology, Therapeutic Intervention, and the Quest for a Vaccine. Toxins [Internet]. 2022 Feb 14;14(2):138. Available from: https://www.mdpi.com/2072-6651/14/2/138

[15] Boix E, Wu YN, Vasandani VM, Saxena SK, Wojciech Ardelt, Ladner JE, et al. Role of the N Terminus in RNase A Homologues: Differences in Catalytic Activity, Ribonuclease Inhibitor Interaction and Cytotoxicity. Journal of Molecular Biology. 1996 Apr 19;257(5):992–1007.

[16] Furiga A, Pierre G, Glories M, Aimar P, Roques C, Causserand C, et al. Effects of Ionic Strength on Bacteriophage MS2 Behavior and Their Implications for the Assessment of Virus Retention by Ultrafiltration Membranes. Applied and Environmental Microbiology [Internet]. 2011 Jan 1 [cited 2022 Jul 11];77(1):229–36. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3019717/

[17] Brié A, Bertrand I, Meo M, Boudaud N, Gantzer C. The Effect of Heat on the Physicochemical Properties of Bacteriophage MS2. Food and Environmental Virology. 2016 Jun 14;8(4):251–61.

[18] Wang C, Liu H. Factors influencing degradation kinetics of mRNAs and half-lives of microRNAs, circRNAs, lncRNAs in blood in vitro using quantitative PCR. Scientific Reports [Internet]. 2022 May 4;12(1):7259. Available from: https://www.nature.com/articles/s41598-022-11339-w

[19] Kotrba P, Ruml T. Surface Display of Metal Fixation Motifs of Bacterial P1-Type ATPases Specifically Promotes Biosorption of Pb2+ by Saccharomyces cerevisiae. Applied and Environmental Microbiology. 2010 Feb 19;76(8):2615–22.

[20] Wolters Kluwer. The bowtie method [Internet]. www.wolterskluwer.com. 2024. Available from: https://www.wolterskluwer.com/en/solutions/enablon/bowtie/expert-insights/barrier-based-risk-management-knowledge-base/the-bowtie-method

[21] American Institute Of Chemical Engineers. Center For Chemical Process Safety. Bow ties in risk management: a concept book for process safety. Hoboken, NJ: John Wiley & Sons, Inc; 2018.

[22] Bhalla N, Jolly P, Formisano N, Estrela P. Introduction to Biosensors. Essays In Biochemistry [Internet]. 2016 Jun 30;60(1):1–8. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986445/

[23] Igem.org. 2025. Available from: https://responsibility.igem.org/safety-policies

[24] ISO. ISO 31000:2018 risk management — guidelines [Internet]. ISO. 2018. Available from: https://www.iso.org/obp/ui/#iso:std:iso:31000:ed-2:v1:en

[25] American Institute Of Chemical Engineers. Center For Chemical Process Safety. Guidelines for hazard evaluation procedures. Hoboken, N.J.: Wiley-Interscience; 2008.

[26] Richmond JY, McKinney RW, Centers For Disease Control (U.S), National Institutes Of Health (U.S). Biosafety in microbiological and biomedical laboratories. Washington: U.S. Government Printing Office; 1993.

[27] Igem.org. 2025. Available from: https://responsibility.igem.org/safety-policies

[28] Canada PHA of. Canadian Biosafety Handbook, Second Edition [Internet]. aem. 2016. Available from: https://www.canada.ca/en/public-health/services/canadian-biosafety-standards-guidelines/handbook-second-edition.html

[29] Canada PHA of. Canadian Biosafety Standard, Third Edition [Internet]. www.canada.ca. 2022. Available from: https://www.canada.ca/en/public-health/services/canadian-biosafety-standards-guidelines/third-edition.html

[30] Lihui X, Zhao J, Skonieczna M, Zhou P, Huang R. Nanoparticles‐induced potential toxicity on human health: Applications, toxicity mechanisms, and evaluation models. MedComm. 2023 Jul 14;4(4).

[31] Bistas E, Sanghavi D. Methylene Blue [Internet]. PubMed. Treasure Island (FL): StatPearls Publishing; 2021. Available from: https://www.ncbi.nlm.nih.gov/books/NBK557593/

[32] Oladoye PO, Ajiboye TO, Omotola EO, Oyewola OJ. Methylene blue dye: Toxicity and potential elimination technology from wastewater. Results in Engineering [Internet]. 2022 Dec 1;16(100678):100678. Available from: https://www.sciencedirect.com/science/article/pii/S2590123022003486

[33] Hazard Communication - Guidance For Hazard Determination | Occupational Safety and Health Administration [Internet]. www.osha.gov. Available from: https://www.osha.gov/hazcom/ghd053107

[34] Government of Canada CC for OH and S. CCOHS: What is a LD50 and LC50? [Internet]. www.ccohs.ca. 2023. Available from: https://www.ccohs.ca/oshanswers/chemicals/ld50.html#section-1-hdr

[35] Sigma‑Aldrich. Safety Data Sheet: Disodium hydrogen phosphate (Product No. NIST2186II) [Internet]. Version 8.3; 09 June 2024 [cited 2025 Oct 5]. Available from: https://www.sigmaaldrich.com/US/en/sds/sial/nist2186ii

[36] Kim E, Yoo S, Ro HY, Han HJ, Baek YW, Eom IC, et al. Aquatic Toxicity Assessment of Phosphate Compounds. Environmental Health and Toxicology [Internet]. 2013 Feb 7;28:e2013002. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3577115/

[37] Sigma-Aldrich. Safety Data Sheet: Sodium chloride (Product No. S7653). Version 6.17, revision date 09 Oct 2025 [Internet]. St. Louis (MO): Sigma-Aldrich Inc.; 2025 [cited 2025 Oct 5]. Available from: https://www.sigmaaldrich.com/US/en/sds/sial/s7653?srsltid=AfmBOorDYR0MZ7qAMgXwGbD_tkIEmPz7hUu1YCpE5tXgYKY53qnUpCtN

[38] Sigma‑Aldrich. Safety Data Sheet: Potassium chloride (Product No. P9333). Version 6.13, revision date 28 Apr 2025 [Internet]. St. Louis (MO): Sigma‑Aldrich Inc.; 2025 [cited 2025 Oct 5]. Available from: https://www.sigmaaldrich.com/US/en/sds/sial/p9333?srsltid=AfmBOopk6wzA2L0Rr6aGoY_hs5VB0LGmaDl8d7ncJbYKJe7_byY‑X2Wf

[39] Sigma‑Aldrich. Safety Data Sheet: Ethyl Alcohol, pure (Product No. 459836). Version 6.17, revision date 07 Aug 2025 [Internet]. St. Louis (MO): Sigma‑Aldrich Inc.; 2025 [cited 2025 Oct 5]. Available from: https://www.sigmaaldrich.com/US/en/sds/sial/459836?userType=undefined

[40] Sigma‑Aldrich. Safety Data Sheet: Sodium hypochlorite solution (Product No. 425044). Version 6.13, revision date 07 Sep 2024 [Internet]. St. Louis (MO): Sigma‑Aldrich Inc.; 2024 [cited 2025 Oct 5]. Available from: https://www.sigmaaldrich.com/US/en/sds/sigald/425044?srsltid=AfmBOopg6OyheuoS1jrbu3b9eaDNiSAPdyTvCH7HCnFboofYqWUs4yCc

[41] PubChem. Calcium Chloride [Internet]. Nih.gov. PubChem; 2024. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Calcium-Chloride#section=Hazard-Classes-and-Categories

[42] Sigma-Aldrich. Safety Data Sheet: Calcium chloride (Product No. C8106). Version 6.6, revision date 03 Mar 2024 [Internet]. St. Louis (MO): Sigma-Aldrich Inc.; 2024 [cited 2025 Oct 5]. Available from: https://www.sigmaaldrich.com/US/en/sds/sial/c8106?userType=undefined&srsltid=AfmBOorlRWhx5Rgsd7fJDpGrvVvmB9cYkqfWI0YhPZYPtQt0FtsF9hO

[43] Sigma-Aldrich. Calcium acetate monohydrate [Internet]. St. Louis (MO): Sigma-Aldrich; 2025 Apr 28 [cited 2025 Oct 5]. Available from: https://www.sigmaaldrich.com/US/en/sds/sigald/402850

[44] SAFETY DATA SHEET [Internet]. 2025 [cited 2025 Oct 6]. Available from: https://www.neb.com/-/media/32c333f1ac19465daec7b65e3fcc76ff.pdf?srsltid=AfmBOopU4nqicDk-5SwKR0CfxUwk6ogwrrRXK7rAUnyZfkAi_89xkfP1

[45] Farrell RE. RNA Methodologies: Laboratory Guide for Isolation and Characterization. Academic Press; 2010.

[46] PubChem. Dithiothreitol [Internet]. Nih.gov. PubChem; 2025 [cited 2025 Oct 6]. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Dithiothreitol#section=Toxicity

[47] Sigma-Aldrich. DL-Dithiothreitol Solution [Internet]. St. Louis (MO): Sigma-Aldrich; 2025 Apr 30 [cited 2025 Oct 5]. Available from: https://www.sigmaaldrich.com/US/en/sds/sigma/646563

[48] Sigma-Aldrich. N-Acetyl-L-cysteine [Internet]. St. Louis (MO): Sigma-Aldrich; 2023 Aug 3 [cited 2025 Oct 5]. Available from: https://www.sigmaaldrich.com/US/en/sds/sial/a7250

[49] PubChem. N-Acetyl-L-Cysteine [Internet]. Nih.gov. PubChem; 2025 [cited 2025 Oct 6]. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Acetylcysteine#section=Mechanism-of-Action

[50] SAFETY DATA SHEET [Internet]. [cited 2025 Oct 6]. Available from: https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/safety-data-sheet/unmodified-dna-oligo.pdf?sfvrsn=2fb83707_17

[51] Sigma-Aldrich. 1-Dodecanol [Internet]. St. Louis (MO): Sigma-Aldrich; 2024 Sep 7 [cited 2025 Oct 5]. Available from: https://www.sigmaaldrich.com/US/de/sds/SIAL/44095

[52] Sigma-Aldrich. Sodium dodecyl sulfate [Internet]. St. Louis (MO): Sigma-Aldrich; 2025 Jul 30 [cited 2025 Oct 5]. Available from: https://www.sigmaaldrich.com/US/en/sds/sial/l4509

[53] Canadian Food Inspection Agency. Overview of how Canada prevents, prepares and responds to bird flu outbreaks - inspection.canada.ca [Internet]. Canada.ca. 2023. Available from: https://inspection.canada.ca/en/animal-health/terrestrial-animals/diseases/reportable/avian-influenza/prevention-preparedness-and-response