Team:UofC Calgary/Safety

iGEM Calgary 2016

Safety

Safety Considerations in the Lab

How we prepared for lab work

All Principal Investigators, mentors, and undergraduate researchers were required to complete lab safety training and safety courses developed by the University of Calgary's Environment Health and Safety (EHS) services prior to working in the lab. These mandatory safety training courses included courses on occupational health and safety, laboratory safety, hazard assessment, incident reporting and investigation, spill response, biosafety, bloodborne pathogens, and an updated versions of the WHMIS course. The courses cover biological containment protocols, handling of hazardous materials such as liquid nitrogen, and disposal of waste, as well as standard safety and laboratory practices. All required us to take a test following each course, which certified safe lab work under the EHS Guidelines. All team members, advisors, and mentors received credit for each course and training program listed, and supervisors were present in the lab at all times to oversee undergraduate work.

The University of Calgary has a university-wide Biosafety Committee, whose guidelines for safe biological laboratory practices were adhered to throughout the project. The team’s lab benches and experimental plans were assessed and deemed safe to proceed with by this Biosafety Committee. The Univerity's Environment Health and Safety (EHS) services provided additional training for individuals working with radiation and irradiated cells.

Our project utilized Bacillus subtilis and a commonly used lab-strain of Escherichia coli, TOP10. Both are non-pathogenic and non-infectious, and are classified as Biosafety Level 1 organisms (BSL-1). Therefore, these organisms posed no significant risk to researchers. Since the BSL-1 cells (E. coli and B. subtilis) have GRAS labelling, the main cloning component of out project did not require ethics approval by review boards. Some team members worked with HCT116 and 1BR3 primary cell lines, which are human colon carcinoma and human skin fibroblast cell lines and are classified as Biosafety Level 2 (BSL-2).The cell lines were received from completely anonymous donors. We handled these cell lines at containment level 2 in accordance with the Bloodborne Pathogens Standard and Biosafety Committee guidelines.

Safety Considerations for the Device

Structure of the Patch

  • The circular design of the packet containing media and B. subtilis spores encourages equal distribution of liquid pressure around the circumference to prevent the patch from breaking open at the seams. The packets are made of a flexible polyethylene material, which is advantageous for distributing external forces against the patch, such as accidental bumping or squishing. This distribution of pressure prevents the material from rupturing due to accumulated forces along seams or corners.
  • The fact that our patch is non-invasive and only present in the external part of the body promotes safety better than an implant.
  • Choosing Patch Materials

  • All materials used in the patch are chemically and biologically compatible, which decreases the risk of an immune response in the user.
  • The backing layer of the patch is made up of a strong, flexible, and gas-permeable material. This allows users to move with ease saves them the worry of the patch tearing apart. This layer also prevents bacteria from escaping.
  • The size controlling membrane is made up of polymers that prevent bacteria from flowing through the patch, but allow our peptide to pass. The bacteria will not come into contact with the skin, which protects the user from the possibility of infection or immune responses.
  • The adhesive chosen to affix the patch to the skin has high oxygen/gas permeability, causes low pain upon removal from sensitive skin, and promotes diffusivity of the drug. The adhesive has been tested by Dow Corning and causes no significant effects on test animals.
  • The release liner is peeled from the adhesive layer and disposed of when the patch is applied to the skin.
  • Considering Human Use

    Advantages of a Patch

  • Using a patch is preferable to many other drug delivery systems. A patch gives the user the freedom to continue a course of medications even with an upset stomach, as the drug will not need to go through the digestive system. It is less invasive than an implant or injection, and delivers the therapeutic agent continuously, meaning the dosage is always above the threshold required for a therapeutic effect, and the user does not need to worry about taking their medication at the same time each day.
  • Possible Problems with the Patch

  • Accidental breakage of the patch while attached to skin may cause infections if the area is not disinfected properly. The risk is increased if there are open wounds on the skin near the application area.
  • Patch may cause severe harm if swallowed.
  • Potential skin irritation may occur. However, our initial mouse trials have shown no signs of inflammation.
  • In Vivo Mouse Trials

  • The safety of the radioprotective patch was assessed by conducting in vivo mouse trails to test the patch, safety of the bacteria and our mBBI peptide. Our mouse trial protocol was followed and approved by the Health Sciences Animal Care Committee (HSACC) who review the ethics concerning animal care and testing at the University of Calgary.
  • Documentation of ethics approval from the animal studies is available upon request as it contains confidential information that can not be made publicly available.
  • Containment

  • Contamination would be an issue if the bacteria were to escape from our patch, as bacteria could infect the user or colonize foreign planets. To protect against the event that our radioprotective patch causes contamination in space or on earth, our patch has several engineering controls in place, such as the protective physical barriers of the patch that would prevent the outbreak of bacteria. The backing layer and the size-controlling membrane prevents the bacteria from escaping outside of the patch and being released onto the skin, respectively.
  • Although our initial diffusion assays have shown that bacteria can diffuse through our semipermeable membrane, we have begun testing with other smaller filters including peptide dialysis membranes and 0.2 micron size filters. We have also shown success with membranes used to filter-sterilize liquids.
  • See pages 15 - 16 of the Device Manual to tackle contamination issues.
  • Future Considerations for Patch Design

    If we can determine a better membrane that prevents the diffusion of the bacteria, we can use a two layer semi permeable system where the first layer prevents the diffusion of the bacteria and a second layer which further filters BBI for diffusion.

    Safe disposal:

  • See pages 11 and 12 of the Device Manual for disposal information.
  • Safety Considerations of Biobrick Parts

  • Public safety: threonine auxotrophy was employed where integrated cassettes containing flanking regions of homology for the B. subtilis thrC  gene were used. This knocked out threonine synthase in our chassis, making it unlikely to survive outside of conditions where theronine is supplied.
  • Environmental safety:  It’s possible that our B. subtilis  transformed with mBBI could outcompete native microbes and potentially disrupt existing ecologies; however, the integrated mBBI genetic construct would not make a significant difference in the bacteria’s survivability outside the lab. Given the nature of our project and safeguards we have implemented, none of our BioBricks confer unsafe risks to individuals nor the environment.
  • Future Considerations

    We would engineer inducible kill switches that could eradicate the bacteria if need be. Additionally, integrating mBBI in various essential genes required for amino acid synthesis could provide more opportunities for auxotrophy, increasing the safety of using B. subtilis in the device.

    Safety Forms

  • About Our Lab
  • About Our Project
  • Final Safety Form
  • iGEM

    iGEM is an international competition promoting synthetic biology as a means to solve social, economic and humanitarian problems around the globe. The iGEM Jamboree is held in Boston annually. In 2016, over 300 teams are competing against each other.

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    Located in Calgary, Alberta, Canada.

    • University of Calgary
    • igem.calgary@gmail.com