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.
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.
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.
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.
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 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.
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.
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.
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).
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.