Team:Toronto/Attributions
Attributions
Over the course of this project, iGEM Toronto has had considerable amount of help to overcome many hurdles. We will be forever in debt to the following individuals and organizations without whom this project would have been difficult to make a success.
Wet Lab
Biozone
- Dr. Sean Caffrey - Providing Lab Space
- Dr. Grant Allen - Chemical Engineering - Providing Lab Space
-
Dr. Elizabeth Edwards
- Providing Lab space - Dr. Radhakrishnan Mahadevan - supervisor
- Endang Susilawati - BioZone Lab manager - Equipment & Biosafety training, Emotional Support
- Dr. Grant Allen - Chemical Engineering - Providing Lab Space
- Dr. Grant Allen - Chemical Engineering - Providing Lab Space
All the biological materials were handled in open bench areas. Escherichia coli (E.coli) was the chassis organism used in our study, which is exempt from the requirements of the NIH Guidelines[3], as a risk group 1 organism[4] . We fused the protein binding domain of LacI (from E.coli) and the light oxygen voltage domain (LOV) of Avena sativa.
We used a CI repressor protein from Escherichia phage lambda in our switch. This sequence was obtained from a peer-reviewed article and has no indication of hazardous impact on eukaryotic cell lines.
The anti-CRISPR protein, AcrIIA4, was obtained from Listeria monocytogenes prophages (risk group 1). The protein has been expressed in both bacterial and human cell lines in literature with no measurable toxicity.
The anti-microbial resistance factors in our project were Standard Kanamycin and Chloramphenicol resistance casettes, which are commonly used in research and pose little health risk.
Our engineered system does not have any risk to humans or the environment since our Cas9 protein only targets metabolic genes like araC in E.Coli as well as reporter genes like GFP or mCherry. Standard decontamination and containment through thermal sterilization and certified waste disposal would also mitigate any potential release in the environment.
Human Practices
In terms of future applications, temporal control of Cas9 activity through our switch could reduce off-target edits and increase the fidelity of genomic integrations through homology-directed repair, which should improve the safety of this technology. Although, from an ethical standpoint, there are several key shareholders including patients, medical practitioners and legislative and executive agencies that could be impacted by human gene editing. Thus, we conducted a systematic analysis of the considerations of each of those groups through interviews and a collaborative podcast series.
References
- Canadian Biosafety Standards and Guidelines - https://www.canada.ca/en/public-health/services/canadian-biosafety-standards-guidelines.html
- Biosafety Team in the Office of Environmental Health and Safety - https://ehs.utoronto.ca/our-services/biosafety/
- NIH Guidelines - https://www.canada.ca/en/public-health/services/laboratory-biosafety-biosecurity/pathogen-safety-data-sheets-risk-assessment.html
- iGEM Risk Groups - https://2017.igem.org/Safety/Risk_Groups
