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From 2011.igem.org

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Safety

As part of the requirements from each iGEM team, we have answered the safety questions in the context of our project.

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1. Would the materials used in your project and/or your final product pose:
a. Risks to the safety and health of team members or others in the lab?
No, we are not using any materials that pose risk to anyone’s safety or health. We are only working with level 1 safety organisms and are not working with any toxic or otherwise dangerous materials. The organisms we are working with are Escherichia coli (E. coli) K12 strains BL21 DE3, DH10B and DH5α. In addition, we are using Bacillus subtilis (B. subtilis) strain 168 and Arabidopsis thaliana (A. thaliana) Columbia wt, DR5:GFP and DR:3VENUS strains. The products that will be produced by our bacteria are also not dangerous to humans.

b. Risks to the safety and health of the general public if released by design or accident?
No, our final product is an engineered E. coli that expresses a plant exudate-responsive chemoreceptor and secretes auxin (indole-3-acetic acid, IAA). We have used harmless E. coli K12 strains, B. subtilis strains and A. thaliana which would not pose any threat to the public if released.

c. Risks to environmental quality if released by design or accident?
No, from consulting Dr Alexandru Milcu, an ecologist, and experts from Syngenta, we have confirmed that secretion of natural indole-3-acetic acid into soil would not pose any large-scale threat to its biodiversity. However, as there is always a given level of uncertainty when dealing with biological systems, we have also designed a plasmid containment device that stops horizontal gene transfer to bacteria in the environment.

d. Risks to security through malicious misuse by individuals, groups or states?
No. The parts we are engineering are not in themselves dangerous. However, it would be conceivable that, if combined with other parts and components, it would be possible to construct a dangerous organism. Nevertheless, this can be said of most synthetic biology parts.

Please explain your responses (whether yes or no) to these questions.

Specifically, are any parts or devices in your project associated with (or known to cause):
- pathogenicity, infectivity, or toxicity?
Yes. However, in isolation, none of the parts that we are using are dangerous. The auxin producing pathway occurs naturally in soil bacteria that are plant pathogens. However, these bacteria use the auxin pathway in combination with virulence factors that are not present in our bacteria. The pathway is also used by plant symbionts and is not inherently dangerous and should not promote pathogenicity to the plants. The chemotaxis receptor we are engineering into our bacteria naturally occurs in Pseudomonas aeruginosa, a pathogen of various organisms including humans and plants. However, the receptor is not in itself dangerous. On the other hand, holin (which is part of our Gene Guard module) is a bactericidial protein that works in conjunction with endolysin. However, it will only be active in bacteria that actively take up our genetically modified plasmid.

- threats to environmental quality?
No. Auxin secretion may favour some plants over others and this may skew the population in affected area. However, we are targeting areas that already have degraded soil and reduced biodiversity. Therefore, we believe that the benefits of our system will far outweigh this potential risk. We are hoping that the application of auxin will eventually increase biodiversity. The auxin should therefore not negatively influence the environment.

- security concerns?
As previously stated, it is conceivable that the parts we are engineering could be used for malicious purposes.

2. If your response to any of the questions above is yes:
a. Explain how you addressed these issues in project design and while conducting laboratory work.
We have addressed the issues by doing various experiments on A. thaliana to see the effects of different auxin concentrations. This will inform our design by telling us how much auxin our bacteria should be able to produce. Furthermore, to avoid horizontal gene transfer, we have come up with a design for a containment module. This module will ensure that if the auxin genes are ever transferred to any bacteria other than our own they will also be transferred with a holin and endolysin gene. These genes will lyse any cells that have obtained the genetically modified plasmid.

b. Describe and document safety, security, health and/or environmental issues as you submit your parts to the Registry.

3. Under what biosafety provisions will / do you operate?
a. Does your institution have its own biosafety rules and if so what are they? Provide a link to them online if possible.
Imperial College has its own biosafety rules. These rules limit the use of infective and GM modified organisms to certified laboratories and state the requirement to get approval for work with them and the requirement to dispose of the organisms appropriately and report any accidental release and accidents to the Health and Safety department. The policy can be found here. Another, more comprehensive outline of how to deal with specific biological agents can be found here.

b. Does your institution have an Institutional Biosafety Committee or equivalent group? If yes, have you discussed your project with them? Describe any concerns or changes that were made based on this review.
Yes, there is a biosafety committee at our institution and we have discussed every aspect of our project with them. Following their advice, we have made modifications and changes required to minimise risk for the team and all other individuals working in the same facility.

c. Will / did you receive any biosafety and/or lab training before beginning your project? If so, describe this training.
Before we were allowed into the lab we received a lab induction. In this induction we were shown the proper methods of waste disposal and were also shown where the fume hoods are and the precautions that should be followed when handling GMOs. We were also given an induction for the plant lab and were told how to handle GM plants. Furthermore we were told where the emergency exits are and the proper assembly point in case of a fire. We received biosafety advice and help with estimating the impact of our project on the environment and society from experts at the LSE BIOS Centre.

d. Does your country have national biosafety regulations or guidelines? If so, provide a link to them online if possible.
There are national biosafety regulations in Britain. The full list can be found here.

4. OPTIONAL QUESTION: Do you have other ideas on how to deal with safety or security issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?
We have designed a novel containment switch. This device is able to kill non-engineered bacteria that take up the genetically engineered plasmid, thereby ensuring containment of the GM plasmid to engineered strains of bacteria.

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