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 complete safety courses developed by Environment Health and Safety (EHS) prior to working in the lab. The mandatory safety training courses included updated versions of the WHMIS course, an occupational health and safety course, a laboratory safety course, a hazard assessment course, an incident reporting and investigation course, a spill response course, a biosafety program course, a biosafety laboratory course, and a biosafety bloodborne pathogens 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 were required to take a test following each course, which certifies safe lab work under EHS Guidelines. All team members, advisors, and mentors received credit for each listed course and training program, 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 the Biosafety Committee. The Environment Health and Safety (EHS) 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). These organisms, therefore, posed no significant risk to researchers. Since these BSL1 cells (E.coli and B.subtilis) have GRAS labelling, our main project did not require ethics approval by the review boards. Some team members worked with HCT116 cell lines and 1BR3 cell lines, which are human colon carcinoma and human skin fibroblast cell lines that 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.
Having a quadrilateral design can cause accumulation of pressure along the edges when a packet is accidentally squeezed. This would potentially rupture the material faster than having distributed pressure along its sides.
The fact that our patch is also 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 is chemically compatible and biocompatible, which poses less risks of user immune response.
Backing layer: The backing layer is made up of strong, flexible, and gas permeable material. This will allow users to move with ease and saving them the worry of the patch tearing apart. This layer also prevents the bacteria from escaping.
Size-controlling membrane: The size controlling membrane is made up of polymers that prevent bacteria from flowing through the patch, only allowing peptide to pass. The bacteria, therefore, will not cause infections as it is shielded from the skin.
Adhesive: The adhesive chosen has high oxygen/gas permeability, causes low pain upon removal to sensitive skin, and promotes diffusivity of the drug. This has been tested by Dow Corning and causes no significant effects on test animals.
Release liner: The release liner is disposed after being peeled.
Considering Human Use
Using a patch gives users freedom to take medication even with an upset stomach, as the drug will not need to go through the digestive system.
Accidental breakage of the patch while attached to skin may cause infections if not disinfected properly.
Patch may cause severe harm if swallowed.
Potential skin irritation may occur. Our initial mouse trials however have shown no signs of inflammation.
The safety of the radioprotective patch was assessed by conducting in vivo mouse trails to test the patch, safety of the bacteria and the protein BBI. The mouse trial protocol was followed and approved by the Health Sciences Animal Care Committee (HSACC) who review animal care and testing.
Documentation of ethics approval from the animal studies: available upon request.
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.
Containment
Contamination issues may be a problem in space if the bacteria were to escape. To protect against the event that our radioprotective patch causes contamination in space or on earth, we would engineer inducible kill switches that could eradicate the bacteria if need be. Additionally, engineering controls such as the protective physical barriers of the patch would prevent the outbreak of bacteria. Various safety mechanisms have been included in the device and they are listed below.
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.
See pages 15 - 16 of the Device Manual to tackle contamination issues.
Safe disposal:
See pages 11 and 12 of the Device Manual for disposal information.
Safety considerations of Biobrick parts
Public safety: Auxotrophy using Threonine was integrated in Bacillus making it unlikely it will survive outside of lab.
Environmental safety: It’s possible our Bacillus incorporated with BBI could possibly outcompete native microbes, however the the bacteria’s survivability outside the lab has not been increased enough to make a significant difference
The engineered organism could potentially disrupt existing ecologies and be difficult to contain; however, as mentioned previously, integrated BBI would not make a difference in the bacteria’s survivability outside the lab, and we have taken care to ensure that the Threonine auxotrophy is fatal to engineered microbes. Namely, 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
A kill switch in Bacillus should be designed as engineering a kill switch into standardized plasmids could be useful for future iGEM competitions. Additionally, integrating BBI in multiple sites would give more auxotrophic sites, increasing the safety of using B.subtilis in the device.
Safety forms:
About Our Lab
About Our Project
Final Safety Form
