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From 2012.igem.org

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SAFETY

Laboratory Safety

The Ribozyme Project is not expected to raise any research, public or environmental safety concerns other than those normally associated with Biosafety Level 2 organisms, such as Escherichia coli (DH5-alpha), which is classified as very low to moderate. The use of this project is primarily reserved for research and laboratory use, therefore, should not purposefully be exposed to the public or environment except after further testing in its specific applications (such as with particular fusion proteins). Furthermore, the basis of our project is to establish a self-excising sequence (ribozymes), which should limit the expression of any intervening sequences to the RNA level. If the intervening sequence were something of environmental or public relevance (such as antibiotic resistance), the experimental design indicates that the sequence will be removed and, thus, not expressed. This is a relevant contribution of the design in limiting expression to the RNA level, which eases environmental hazard concern upon the accidental release of a GMO containing this biobrick. Therefore, the new biobrick parts submitted should not raise any safety issues.

The necessary facility, equipment and handling procedures associated with Level 2 Biosafety concerns were met:

1.Pipetting aids
2.Biosafety cabinets where applicable
3.Laboratory separated from other activities
4.Biohazard sign
5.Proper safety and disposal equipment, including autoclave
6.Personal protective equipment, worn only in the laboratory
7.Screw-capped tubes and bottles
8.Plastic disposable pasteur pipettes, when necessary

All precautions with respect to recombinant DNA were observed:
1.All waste was autoclaved before being thrown away.
2.Researchers practiced aseptic technique and personal hygiene and safety precautions
3.Procedures likely to generate aerosols are performed in a biosafety cabinet
4.Bench surfaces were disinfected with ethanol.
4.Potentially contaminated waste is separated from general waste

Safety Questions

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

• researcher safety
• public safety
• environmental safety?

Researcher safety when using E.coli is slightly increased due to the introduction of recombinant DNA which confers antibiotic resistance. While, the E.coli strain used was relatively harmless, treatment of possible infections may potentially be affected by the antibiotic resistance. Further more, spontaneous mutations which result in increased infectivity may result. However, measures and precautions listed above were taken to minimize even the slight chance infection.

Public safety concerns arose from ideas behind ideas for applications of this project in making pesticides which can overcome insect resistance. The concern arises from the fact that this particular application would be used in the food industry, where members of the public may be exposed to genetically engineered organisms and recombinant DNA. The current project however, did not involve any implementation of this idea. Any applications of this project would of course be subject to extensive testing, approval by regulatory boards and committees, and would comply with Canadian and international laws to ensure public safety.

Environmental safety is also an issue whenever genes conferring antibiotic resistance are used. The risk of horizontal gene transfer can be substantial so intense testing and experimentation under controlled settings to assess the effects and degree of gene transfer would be required for such applications. Apart from concerns of recombinant DNA being transferred to other organisms, applications involving pesticide production is also an issue as this application involves release of genetically engineered organisms into the environment. While such an application may initially work to kill resistant pests, there would be a fear of further increases in pesticide resistance in future generations. Thus, further testing in controlled conditions would also be required to assess the extent of this possibility.

2. 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?

This project utilized the same parts from the Waterloo’s 2011 project, and did not raise any a dditional safety issues. While applications of the project would raise safety issues, our proof of concept construction using GFP is relatively safe.

3. 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?

The laboratories operating at the University of Waterloo have obtained permits from the Bio-Safety Committee in order to perform intended research. Since the Waterloo iGEM team performs all laboratory work in a parent lab under the guidance of the Masters and PhD students of that lab, the projects carried out in the lab are covered by the permits obtained by the parent lab.