Safety Questions

  1. Would any of your projet ideas raise safety issues in terms of:
    • researcher safety,
    • public safety, or
    • environmental safety?

  2.           The UofT iGEM team used two bacterial, one fungal, and one plant species in the derivation and assembly of the genetic construct: Escherichia coli 'DH5alpha' (BSL-1), Agrobacterium tumefaciens (BSL-1), Aspergillus niger 'ATCC9029' (BSL-2), and Daucus carota (BSL-1). The genetic constructs were expressed in Arabidopsis thaliana , a model plant organism that has been researched and used extensively in botanical laboratories.
              The E. coli strain DH5alpha is non-pathogenic 1, 2, and as such, it poses minimal risk to the safety and health of the general public if the constructs (or the bacteria) were to be released into the environment. Needless to say, however, stringent sterilization protocols were followed everytime it was used. Similarly, the A.tumefaciens is non-pathogenic and poses no threat to human and environmental health. Even upon escape, it was unlikely to infect other plants as the promoters of the construct were designed specifically for A. thaliana. Again, great care was taken to ensure that such an event did not occur.
              For Aspergillus niger , BSL-2 safety protocols were followed to ensure proper handling and disposal of all media and A. niger used. It was used to clone the phytase gene required for one of the two constructs. If spores are inhaled in large quantities, a condition called aspergillosis results (usually only in immunocompromised patients) 3 . Nonetheless, a policy of handling A. niger under a laminar flow hood while donning a surgical mask was enforced. All media and cultures involved with this fungus were disposed of by a licensed chemical waste disposal service for the University of Toronto's Medical Science Building, in which the lab is housed. The release of A. niger by accident would not be a major concern, despite its BSL-2 status, as it is a fairly ubiquitous fungus found on fruits and vegetables and is consumed daily. The possibility of aspergilloma would likely only result if the fungus was released in very confined spaces with immunocompromised patients; its release poses little to no risk in well-ventilated areas.
              D. carota , the common carrot, does not pose any risk to research, public or environmental safety. It was used to extract the extensin signal for root excretion. The specimen used was bought from the local grocery store, and care was taken to ensure it did not, by accident, end in someone's salad.

              For the first ten days of lab time, our summer research team undergoes a "crash course" in lab training given by a senior iGEM student with at least two years' prior lab experience. We ensure that, once our construct has been designed, each team member reads over all required pathogen safety data sheets (if the organisms in question are BSL-2), and materials safety data sheets for all hazardous compounds we must use in the lab over the course of the summer. This is designed to ensure familiarity with handling and disposable protocols. This "crash course" also teaches all research students essential lab skills, such as: the safe preparation of stock solutions; the autoclaving of all wastes prior to disposal; the proper handling of EtBr; the protocol for agarose gel electrophoresis; safe imaging of agarose gels; proper inoculation technique; transformation, ligation, and digestion; as well as PCR, blotting, and miniprep techniques.

              None of the lab equipment or chemicals pose any serious risk to the researchers or the public. The team instituted a policy involving mandatory use of an "MSDS Binder" for reference (see question 4 below). Whenever possible, all protocols were performed under the fume hood while donning a surgical mask, eye protection, a lab coat, and nitrile gloves. Moreover, majority of lab work was also done in partners (to further increase researcher safety).

  3. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes,
    • did you document these issues in the Registry?
    • how did you manage to handle the safety issue?
    • How could other teams learn from your experience?

  4.           None of the new BioBrick parts (or devices) raise any safety issues and are derived completely from non-pathogenic, non-hazardous species. All cloned and amplified genetic parts were derived from BSL-1 species, with exception of the phytase protein. Despite having been isolated fromA. niger, the physical gene does not pose any threat to human health or environmental safety. In fact, the protein product of this gene has been used in animal feed as a feed additive (to increase phosophrous absorption) 4 .
              No parts or devices in our project are associated with the conferral of pathogenicity, toxicity, or potential infectivity.
              In addition, no parts or devices in our project are associated with any known threats to environmental quality, and utmost care was taken to ensure complete biocontainment and isolation of plant seeds (click here to see our new approach to Botanical Research safety).
              Furthermore, no parts or devices in our project are associated with any known security concerns.

  5. Is there a local biosafety group, committee, or review board at your institution?
    • If yes, what does your local biosafety group think about your project?
    • If no, which specific biosafety rules or guidelines do you have to consider in your country?

  6.           The University of Toronto has an Institutional Biosafety Committee, alongside local biosafety coordinators for each building in which laboratory research is undertaken. As such, the Medical Sciences Building (in which our lab is located), its occupants, the public, or committee members may raise any biosafety concerns and issues with either the local biosafety coordinator, or the campus-wide Biosafety Committee. No concerns or changes to our project, however, were raised.

              Our lab works under BSL-1 provisions, as outlined by the government of Canada's Public Health Agency guidelines on Laboratory Safety, Third Edition (2004) , as governed by University of Toronto. The UofT iGEM team at all times adhered to the policy and procedural framework designed by the Biosafety Committee to ensure that the research was being conducted safely and in conformity with the relevant Acts and regulations.

              The University of Toronto does have its own Biosafety policy, which was last updated in 2007 and is available here. The University of Toronto requires that every laboratory operating under BSL-1 conditions or higher must apply annually (if BSL-2 or higher) or biennially (BSL-1) for a biosafety certificate, and are subject to inspection by the University's Biosafety committee at any time during the validity of said certificate. Importation permits are not required by the University or the Government of Canada for the handling of BSL-1 organisms such as E. coli strain DH5alpha.

  7. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

  8.           The UofT iGEM team has instituted a policy involving mandatory use of an "MSDS Binder" in every iGEM lab, in which Material Safety Data Sheets may be kept, along with Pathogen Safety Data Sheets for easy reference. By having this information at hand, it will eliminate the need for second guessing with regards to chemical or pathogen disposal and/or handling protocols.

              For future plant projects, the UofT iGEM team would like to recommend (and is currently investigating) three possible approahces to gene flow mitigation in genetically engineered plants. The first possibility is placing the modified DNA in plastids and as such it cannot be transmitted by pollen (in species where plastids are maternally transmitted) 5 . The second possibility is using cytoplasmic male sterile plants (which do not produce viable pollen). The third possibility, very much related to the second, is using cleistogamous plants (i.e. plants whose flowers do not open or open partly).

              More importantly, however, the UofT iGEM Team has created a new human practices (safety) approach, entitled "Biosafety in Botanical Laboratories." There is a document here (available also as a link through the Human Practices webpage) that outlines and details this new approach. Similar to the Biosafety Level (BSL) standards, this "Biosafety in Botanical Laboratories" recommends practices, and precautions that will allow for safe and ethical conduct of work in botanical laboratories from an environmental, and a public perspective.
             In particular, this standard aims to serve the gap left by Biosafety Level (BSL) standards with regards to isolation and biocontainment of plant seeds within a lab. In other words, this new approach aims to prevent of escape of genetically engineered plants (that are potentially invasive or capable of horizontal gene transfer) from a laboratory setting. This safety system would also establish a security net for anyone deciding to take genetically engineered plants to a largescale production level, improving their overall acceptance, with both the farmers and the public. As such this system would not only address bio-safety and bio-security, but also ethics.
              This fulfills one of the three requirements for the gold medal.

    Sources Cited

    1 Taylor RG et al. (1993) E. coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing. Nucleic Acids Res 21: 1677-8.
    2 Chart H, Smith HR, La Ragione RM, et al. 2000. An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5alpha, and EQ1. J Appl Microbiol. 89(6): 1048-1058.
    3 Zmeili OS, Soubani AO. 2007. Pulmonary aspergillosis: a clinical update. Q J Med. 100:317-334.
    4 Kim T, Mullaney EJ, Porres JM, Roneker KR, Crowe S, Rice S, Ko T, Ullah AH, Daly CB, Welch R, Lei XG (2006). "Shifting the pH profile of Aspergillus niger PhyA phytase to match the stomach pH enhances its effectiveness as an animal feed additive". Appl Environ Microbiol 72 (6): 4397-4403.
    5 Bock, Ralph; Karcher, D; Bock, R (2007), "Determining the transgene containment level provided by chloroplast transformation"
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