Team:OLS Canmore/Safety

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SAFETY

The iGEM team of Our Lady of the Snows Catholic Academy has considered and implemented various safety practices in order to protect our team, students, staff, and world around us. As we have taken safety considerations into account, we are are confident that our final product will have an overall beneficial effect. Our project will prove to be beneficial for those in municipalities who are dealing with the buildup of hair in local wastewater treatment facilities, as a more efficient method to rid of these clogs would present itself through our construct. The poultry industry would also benefit from our project because it will allow for the elimination of feather waste, while also creating useful byproducts. However, these benefits are only applicable if our construct is safe. As we continue to practice safety in the lab and lab safety education, we are optimistic that we will create a low-risk construct that would be suitable for proper implementation in wastewater treatment facilities as well as in the poultry industry, both on a local and global scale.

This year, our team has faced two main safety considerations. The first of these is that in the future, we hope to implement our construct into municipal water systems and farm environments, thus we had to consider and develop an implementation plan for our project. Our second concern is centered around the location of our lab. Our school ranges from grades pre-K to 12 and in addition to our own students, we also share our facilities with our neighbouring school. Our lab is situated just off the main science lab and when unlocked, it is accessible to all students and staff members of both schools. Due to these two important factors, it is vital that we take safety into consideration, both in and out of a lab setting.

The safe implementation of our project is a large concern for our team, as our community and the area surrounding it will be directly affected. Our construct is being designed for application into wastewater treatment facilities, as well as poultry farms and rendering plants. Due to the fact that these companies are vital and affect a large group of people, it is of great importance that our project will not cause harm in any way. Thus, we made it a goal of this year to create an implementation plan. Currently, we are delving into different methods to prevent the exposure of our construct into the environment, general water source, and the general public. Being situated in a wildlife corridor outside of a national park makes the safety of the environment one of our main priorities. Containment is necessary as we do not want to cause potential harm to our animals or ecosystem as a whole through contamination of water. Our team has done extensive research on safe ways to contain our enzyme that would be easy to implement on a mass scale. Through this research, we discovered bioreactors.

To put simply, a bioreactor is a closed container that is used on an industrial level, most commonly in wastewater treatment facilities, to break down biological material. Many bioreactors are self-cleaning, difficult to break, and most importantly, safe (1).

Utilizing a bioreactor would allow for safe and easy integration of our system in both the poultry industry and the waste water treatment industry. In the poultry industry, large quantities of feathers could be “dumped” into the bioreactor and from there would be left to break down. The limited contact of the bioreactor with the workers would ensure that it is safe, contains the construct, and provides the optimal conditions necessary for our construct to work. In wastewater treatment plants, the situation would be very similar, however we would have to integrate our construct into an already existing system. Because of this, we are currently working on the development of a safe solution within the waste waste treatment industry, as their system is more intricate and hair affects the entire system, from toilet to transport.

However, like everything, bioreactors of all kinds can pose threats. If an individual has the proper training required to operate a bioreactor, the chances of injury, problems, or death, are reduced. The most common safety concerns include:

• Bioreactors can be explosive if incompatible chemicals are placed in or around bioreactors.


• Bioreactors generate carbon dioxide if one is in an enclosed space with a bioreactor they can experience oxygen deprivation (only in certain situations where carbon dioxide is produced).


• Clothing that is baggy can be easily caught in bioreactor or other equipment associated with bioreactors.


• Workers may be exposed to waste contaminants by inhalation, ingestion or absorption.


• Biological activity of the bioreactors may be enhanced with the addition of nutrients or other chemical agents. These agents may include nutrients, methanol, or other chemicals for pH adjustment (e.g. acids and bases). Workers may be exposed to these chemicals during their application either as a powder or in a liquid state.


• Bioreactors may expose workers to pathogenic microbes during operation and maintenance. However, exposure to these pathogens is usually not a significant concern unless the wastes being fed into the reactors contain pathogenic agents. If the bioreactors are equipped with open aerators, microbe-entrained mists may become airborne. Inhalation of pathogenic microbes may cause allergic reactions or illness. During sludge handling activities, workers' hands may be exposed to microbes and result in accidental ingestion of pathogenic material (1).
  
  

This year, our team is in the beginning stage of building our own prototype bioreactor for experimentation purposes. Because we are only in the construction phase at this time, we have not yet had to implement important safety precautions regarding the bioreactor. This will change however, once our bioreactor is ready for the containment of a true biological system

There are three main steps in regards to safety that we follow in the lab; substitution, administration, and engineering. An example of the substitution step is the substitution of nucleic acid stains. Staining is a technique used in science in order to view DNA in agarose gel electrophoresis. A commonly used nucleic acid stain is ethidium bromide. Although ethidium bromide is a highly sensitive stain that produces good results in agarose gels, it is notorious for its toxicity. The chemical’s MSDS documents state that it is harmful if swallowed and is very toxic if inhaled. It can also irritate the eyes, respiratory system and skin and has the risk of other irreversible effects. Therefore, this stain poses a major safety issue for those who come in contact with it and is an environmental hazard during disposal (2). Because safety is a priority of our team, we opted to utilize a different chemical for staining, called RedSafe. RedSafe is the relatively new and safe alternative to ethidium bromide. The staining protocol and sensitivity of RedSafe is very similar to ethidium bromide, however it is not classified as hazardous waste and can be disposed of according to standard lab procedures (3).
Our administration step of safety includes proper safety training for all science teachers and iGEM team members, wearing personal protective gear, such as lab coats, safety goggles, and gloves. Protective gear is very necessary for working in the lab and lessens the risks associated with it. The precautions we take for the safety of everyone are simple, yet very effective. These precautions include locking the doors of the lab at all times, washing our hands after the completion of lab work, and tying hair back to avoid contamination. All of our equipment is sterilized prior to working in the lab as well as after, and 70% ethanol is used to clean our lab counters after use. We also have additional safety protocols that we practice, as we have limited waste disposal methods in our lab. Due to a considerably low budget, the majority of our equipment is either second-hand or Do-It-Yourself. Previously, our team had undergone certain education to become knowledgeable on lab safety including; lab safety training, Workplace Hazardous Materials Information System (WHMIS), Material Safety Data Sheets (MSDS), aseptic technique, proper sterilization techniques, and biosafety and wet-lab safety protocols. Though not complex, these safety precautions are crucial to the elimination of risks associated with our project. Said risks that our project currently poses are bacterial contamination, contact with acids, nucleic acid stains, restriction enzymes, or Keratinase, as well as burns that can be caused by a flame, warm glass, or metal. In order to protect people against these threats, we use the fume hood for protocols involved with flaming, use the "dremelfuge" (Dremel tool centrifuge) behind a metal barrier, and share understand how to use our equipment safely. This is an example of our engineering step of safety.

Though we take basic lab safety precautions with great seriousness, we must also consider some precautions in regards to the creation of our construct and the form of bacteria we are utilizing. For the basic construction of our project, we have used an IPTG inducible promoter, however during actual implementation, we plan to replace this with a different promoter that is more suited for a full-scale model. By putting a different inducible promoter into our construct, we are making it safe for implementation and are creating a cost-effective and efficient method, as IPTG is very costly and will not be ideal for wide-scale implementation. We have also decided to implement a strong kill switch into our construct, allowing for safety in the case that the enzyme were to negatively affect the environment or denature. When we think about the application of our project, there are many variables to consider, and addressing all of these variables will require an extensive amount of planning, research, and years of development.

Chassis-wise, we are utilizing the K-12 strain of E. coli, which is a genetically modified bacterium that is non-pathogenic (4). Though some strains of E. coli are dangerous and can cause sickness, the strain we are using poses no threat to human life. Thus, the amount of safety precaution we must take to prevent dangers related to our chassis is lessened. This chassis has a low probability of survival outside of lab conditions and we do not plan to experiment with anything other than this non-pathogenic strain of E. coli. Our team has grown our bacteria on plates that contain antibiotics such as ampicillin and chloramphenicol. To ensure that our bacteria grows to be what is needed and stays safely inside the petri dish, we have also placed a resistance to these antibiotics within our construct.

Our team has performed two assays in order to test the effectiveness of our project: a dry hair assay and a skim milk plate assay. These assays were performed on real hair and feathers, meaning we had to take certain safety precautions into consideration. Basic lab safety precautions are a given, however some precautions are more complex, such as learning how to deal with the byproducts of the reaction, how to extract them safely and securely, and in which conditions we should perform these tests in order to keep our school safe from any product that may be given off by our assays. We have maintained our safety standards while completing these assays and the safety of those around us is of main priority.

To conclude, our team has taken many different safety factors into consideration as we move forward with our project. We ensure that safety precautions are met in and out of the lab space, as they are critical to the current and next steps of our project.


Citations:

1. Chapter 14 Bioreactors. (n.d.). Retrieved October 14, 2016, from http://www.frtr.gov/matrix2/health_safety/chapter_14.html
2. @. (2013). A toxic death for ethidium bromide. Retrieved October 14, 2016, from http://www.labnews.co.uk/features/a-toxic-death-for-ethidium-bromide-08-10-2006/
3. RedSafe DNA Stain (20,000 X). (n.d.). Retrieved October 14, 2016, from
   http://www.chembio.co.uk/red-safe-dna-stain-20-000-x.html
4. Www.design-to-use.de, T. K. (n.d.). Biosafety. Retrieved October 14, 2016, from http://www.bats.ch/bats/publikationen/1996-1_e.coli/96-1_e-coli_k12.php
   
   
   

Contact us at:
https://www.facebook.com/OLeSsence/
@igem_canmore
larvisais@redeemer.ab.ca