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Would any of your project ideas raise safety issues in terms of:

Researcher Safety,

Nicotiana tabacum - No. NT cells are common cell lines used for decades in plant research. No known hazards have been associated with NT cell research. Reporter genes – No. Fluorescent proteins are a staple of molecular biology, and no known hazards have been associated with their use. Our promoters also pose no threat.

Public Safety,

Nicotiana tabacum - No. We do not intend on developing the project in any way such that the public would encounter our project. Even so, in a hypothetical commercial development, NT cells are not expected to put the public at risk. NT cells are not viable outside a nutrient-rich environment.

None of the parts we intend to make, promoters or fluorescent reporters, have shown any health risks to date.

Environmental Safety

- No. While other plant models could conceivably cross with wild-type plants and generate unforeseeable hybrids, NT cells mitigate that risk. Because NT cells are incapable of sexual reproduction and can only proliferate through clonal propagation in their nutrient-rich media. Containment of the cells is easier to manage with less risk should the NT cells ever breach containment. Therefore, we would not expect any of our promoters or reporter genes to reach the environment. We are ensuring proper containment of transformed Agrobacterium.

Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?

- No. Our reporter genes are standard fluorescent proteins used repeatedly not just in iGEM but research abroad. Our modified plasmid poses no risk to the researchers, public, or environment. The inducible promoters selected are found naturally in plants and are predicted to pose no risk.

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?

- Yes. The University of Nevada, Reno Institutional Biosafety Committee supports our project, especially with regard to the fact every member of the iGEM team completed training on NIH Guidelines on Recombinant Organisms. An additional safety course conducted by the University of Nevada, Reno Environmental Health & Safety Department trained each member in the certified Lab Safety Workshop on chemical safety, biological safety, chemical hygiene, ventilation, and waste management. Therefore, all iGEM members have been trained in the handling and disposal of transgenic bacteria.

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?

The steps previously and currently taken by iGEM teams to design systems where modified organisms can be screened or terminated under certain conditions are excellent ways of providing safety. Certainly, controlling where and how organisms can grow helps to ensure modified organisms do not unintentionally interact with the environment. These are all internal controls, and while 99.9% reliable, we believe the risk of unforeseen mutation cannot be overlooked. Several systems of control will have a few layers of regulation, but there could likely be one nexus of vulnerability that the whole system hinges upon, and the question must be asked what happens if that node of control fails? A dysfunctional fluorescent protein may not deserve attention, but genes regulating proliferation, reproduction, or repressors could cause bacteria, fungi, or plants to escape containment. Adding layers of regulation do help, but ultimately they affect the quality of the plasmids or genes used and may ultimately hinder the goals set for the project.

Therefore, we propose one additional way of providing safety in an iGEM doomsday scenario to help in the event of some future iGEM bacteria that infects humans or plants, affecting public health or crops. Perhaps, as iGEM grows and commercial applications become apparent, for plant projects, iGEM could require a constitutively expressed fluorescent protein alongside any novel gene to track the plants. iGEM-compatible internal controls have been designed, for example Harvard’s project, but in the rare event those mechanisms fail, a constitutively expressed fluorescent protein would be an easily identifiable way of tracking iGEM plants.



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