Why Safety is Important
          Upon entering a lab there are certain risks one is exposed to, such as reactive chemicals and equipment, which is why it is crucial to take preventative measures. Daily safety procedures for safe wet lab work to be conducted include proper dress attire which entails a lab coat, closed-toe shoes as well as long pants to protect your legs and skin from the potential dangers. It is also important to ensure long hair is to be tied back and loose jewelry is not worn to prevent potential contamination of the materials you are working with. Another tactic to be used to fight against contamination (especially when working with RNA in our project) is the use of ethanol sprays, as it is effective in killing bacteria, microbes, and other microorganisms. Some materials that are regularly cleaned with ethanol solution are pipettes, workbenches, gloves, all gel casting trays. Cleaning work areas are always important, for example, when our team conducts in vitro transcriptions, we always emphasize and prioritize a clean work area that has been thoroughly wiped down and sterilized with ethanol sprays and RNAzap to prevent RNase contaminations.
 



 Protocols 
          Apart from lab dress codes, it was also critical that every member of our team received proper WHMIS training and certification prior to conducting experiments in the lab. Members are also always accompanied by either an advisor or our primary investigator when conducting experiments in the lab.
    
 Dual Use Considerations 
          Our team has also looked into Dual Use Research, which is research into how every scientific development and project can be used for both ethical and unethical purposes (Wimmer, 2018). Additionally, we attended a workshop held by SynBioCanada on dual use and its importance in synthetic biology research. 
 
          Dual Use Research can be applied to Dia-Beatable in two ways. Our project utilizes self-amplifying RNA to produce the insulin gene in the human body. However, self-amplifying RNA could also be used to amplify harmful genes such as those in diseases. Furthermore, self-amplifying RNA could be maliciously utilized to amplify too much of a gene to purposefully cause harm to an individual’s body and health. For example, over-amplifying the enzyme, cholinesterase could result in the body not being able to contract muscles and nerves. As cholinesterase breaks down the molecule, acetylcholine which is responsible for contracting muscles and nerves (Korabecny & Soukup, 2021). Even over production of insulin is dangerous (Kolb, et al., 2020). 
 
          Malicious use of Dia-Beatable is prevented by our design, our team, and gene distribution screening protocols. An essential component of Dia-Beatable is a molecular switch for controlling insulin production within a cell (see parts and engineering page for more details). We submitted a check-in form to the iGEM Safety &Security Committee which was approved July 15th, 2022, with the special note “Team’s work can go ahead as planned, but I strongly recommend performing tests in mammalian cells only when a robust proof-of-concept has been achieved”. Before ordering sequences we discussed controls, containment, and experimental techniques. Our team is trained in safe practices and knows that use of all project parts is restricted to wet labs where it can be monitored. An additional level of security comes from companies such as IDT who perform high-level screening of sequences to ensure products being synthesized are not from export controlled species. Even with our good intentions, we were unable to acquire Venezualan Equine Encephalitis Virus (VEEV) sequences and focused our wet lab work solely on optimizing the RNA aptamer control mechanism. VEEV may be a controlled species despite not being part of a toxin because self-amplifying sequences can replicate uncontrollably, leading to higher likelihood of mutations with unknown consequences (Wimmer, 2018).


 
 References 
[1] Wimmer, E. (2018). Synthetic Biology, Dual Use Research, and Possibilities for Control. Defence Against Bioterrorism, 7–11. https://doi.org/10.1007/978-94-024-1263-5_2 

[2] Korabecny, J., & Soukup, O. (2021, July 30). Cholinesterase Research. Biomolecules, 11(8), 1121. https://doi.org/10.3390/biom11081121 

[3] Kolb, H., Kempf, K., Röhling, M. et al. Insulin: too much of a good thing is bad. BMC Med 18, 224 (2020). https://doi.org/10.1186/s12916-020-01688-6