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Safety

iGEM Toronto is working towards a method of developing an on-site DNA extraction kit to help general stakeholders test for the presence of Oak Wilt, in a manner that is safe for everyone. The team adheres to safety precautions which allows us to handle the organic materials and perform the required lab techniques.

Laboratory Safety


iGEM Toronto team is in full compliance with the safety and security requirements of the iGEM competition. The lab experiments were conducted in the University of Toronto Biomedical Engineering Teaching Lab, Dr. Keith Pardee’s lab and Biozone - Centre for Applied Bioscience and Bioengineering Research Lab. All laboratories fall under biosafety level 1 and level 2.

Before we start our work in the lab, all team members are required to complete EHS601 Laboratory Biosafety Training (https://ehs.utoronto.ca/our-services/biosafety/biosafety-training/) and pass the exams. In addition to the mandatory training by the University of Toronto Environmental Health & Safety Department, our lab members completed lab-specific biosafety training, general lab safety and equipment training. At all times, our lab supervisor Gary Hoang (BME Teaching Lab) or Ethan Agena (Biozone) or Jennifer Doucet (Dr. Pardee’s lab) was present when our lab members were in the laboratory.

Project Safety


NEB Master Mix + Fluorescence Dye

The Warmstart LAMP Master Mix is a solution for Loop-Mediated Isothermal Amplification (LAMP) of DNA or RNA (RT-LAMP) targets and provides clear visual detection of amplification (a solution change from pink to yellow). This mixture is classified as not hazardous according to regulation (EC) 1272/2008 [CLP]. The tubes containing the master mix are disposed into the biohazard bin after use. Proper lab protective equipment was used when handling this master mix.

The product has no adverse effect on the environment when disposed of in accordance with local environmental legislation and will not be of harm to the stakeholders (i.e. the users) when used with the proper care.


Lucigen Master Mix

The Lucigen LAMP Master Mix is a The LavaLAMP DNA Master Mix greatly simplifies reaction optimisation by limiting optimization to target specific components/conditions such as LAMP primer design, target concentration and reaction temperature. Additionally, this master mix is heat stable at 90 °C for ≤5 minutes, which enables the addition of a reaction preheating step which may, depending on the target, increase assay sensitivity and decrease time to results.
This mixture is classified as not hazardous according to regulation. The tubes containing the master mix are disposed into the biohazard bin after use.


DNA Fragment

According to the Canadian Food Inspection Agency (CFIA), a permit is required for us to import the whole genome of B.fagacearum into Canada. Under “D-99-03: Phytosanity import requirements to prevent the entry of Oak Wilt Disease from the Continental United States”, B.fagacearum is considered an invasive species, and a permit is required in accordance to the Plant Protection Act.

Our team thinks that the import of the whole genome is not a good decision, as it may potentially cause a spread of an invasive species that never occurred in Canada. More than that, the time and cost for us to get a permit was not affordable as well. As the genome is a fungus, it may form spores, which may even result in contamination of the lab.

Therefore, we decided to only experiment on certain segments of interest of the genome, as we are only targeting the specific regions with the LAMP method. We do not need the whole genome to make our experiment work, and this will also eliminate the risk of leakage and contamination, as well as the extra cost of money and time.

Waste from the DNA fragments, such as supernatants from centrifugation steps, is to be considered potentially infectious. Before disposal, the waste must be autoclaved or incinerated to destroy any infectious material. The disposal followed the official regulations.


LAMP primers

LAMP primers are synthetic products produced by IDT. The product does not contain substances which are considered to be hazardous to health, as long as the lab members handling the primers follow the regular biosafety procedures. The tubes containing the primers are disposed into the biohazard bin after use.


Dipsticks DNA Extraction Kit

The product does not contain ingredients which are considered to be hazardous to health, as long as the lab members handling the Dipstick DNA Extraction Kit follow the regular biosafety procedures. The sample, the stick, and the extraction solution are disposed into the biohazard bin after use.


Freeze Drying

The freeze-drying procedures were executed by Jennifer (lab supervisor in the Pardee’s Lab). Jennifer operated the freeze-dryer and handled our materials with caution. Although the lab members were not the operator of this activity, we were informed of the hazards associated with freeze-drying. First, there is a risk of cold burns as we pre-freeze the samples in liquid nitrogen. Second, there is a risk of implosion of glassware as we vacuum the sample in the glass vessels. Third, there is a risk of burns from the refrigeration chamber as we take the sample in and out.


Handling Cladosporium cladosporioides (CL-1) fungus

CL-1 fungus was used to verify that the Dipstick DNA extraction tool works on samples that contain fungus. It is a biosafety level 1, non hazardous substance according to regulation 29 CFR 1910/1200. The experiment was performed under close-supervision of Dr. Yan Wang in the Wang Lab (Specialized lab in fungal genomics and evolution).


Device Safety


This year, iGEM Toronto designed a user-friendly and portable tree xylem sample extraction drill. Our stakeholders will operate the drill in the field. Safety of the drill was under our consideration when we were designing it. The drill is a sharp object, it poses an inherent risk of injury. Therefore, it must be carried inside a box until it is used. Once the tree sample is collected and transferred to our Dipstick DNA extraction tool, the drill will be sterilized using 70% isopropyl alcohol in a device that looks like a dog paw cleaner.

iGEM Toronto designed a liquid mixing device to enable Dipstick DNA Extraction to be performed on site. Since all the samples and reagents are contained in the glassware, it must be disposed into the biohazard sharp container. Proper disposal of the container is required.

The last component of iGEM Toronto’s on site diagnostic tool for oak wilt disease is the heater. The heater can heat up to 100°C, which poses a risk of burning. The heater contains electrical components and batteries, which poses a risk of minor electric shock or burn. Although we were not able to implement the safety design in our proof of concept, we would like to show that the safety risks were under our consideration. We proposed insulating the heating element and electrical energy source from the stakeholder’s hand by boxing our device with some sort of silicon materials.