Team:Edinburgh/Safety

Safety

Before proceeding with our feasibility study, it was important to ensure that the study itself would not cause any harm. Happily, iGEM teams are required to answer the following safety questions.

General safety

Would the materials used in your project and/or your final product pose:

• Risks to the safety and health of team members or others in the lab?
• Risks to the safety and health of the general public if released by design or accident?

No to both.

This year's project involves catalysing reactions by using enzymes displayed on bacteriophage and/or on the bacterial cell surface via Ice Nucleation Protein. A simple test system will involve amylase or GFP; a more complex system would incorporate multiple types of cellulase. None of these things are harmful to humans. There is no pathogenicity, infectivity, or toxicity.

The lab does not use ethidium bromide. The work requires only reagents that are normal for a microbiology lab.

• Risks to environmental quality if released by design or accident?

No. In both the phage and cell display systems, release would cause no harm.

While the full form of Ice Nucleation Protein is apparently used by Pseudomonas syringae to help it attack plants, the version we will use ([http://partsregistry.org/Part:BBa_K265008 BBa_K265008] by UC Davis 2009) lacks the extensive central domains which actually cause ice nucleation; therefore as it stands it has no pathogenicity, and is merely used as a carrier to move fused enzymes to the cell surface.

The phage system is such that BioBrick DNA will not be packaged into the phage, so transfer of DNA to other organisms by this route is impossible. Any escaping phage will contain only normal M13 DNA, and code for normal M13 proteins in the second generation.

Any E. coli that escape (from either system) will have greatly reduced fitness. Our lab strain of E. coli, JM109, is a "disabled" strain, which has very poor survival in the wild.

Therefore, the only plausible way any part of our project could survive in the wild is if horizontal gene transfer moved DNA into other organisms. However, even if this occurred, it could only become established in the metagenome if it confers a fitness advantage to some host. However, extracellular display of enzymes such as cellulases has already been accomplished by evolution, and indeed natural cellulosome systems are almost certainly far superior to our system, a fact which greatly decreases the likelihood of our own constructs conferring a fitness advantage.

In short, our system will enable the use of a cellulosome-like system in E. coli. It will not, however, be a superior system to those that exist in nature in other organisms.

• Risks to security through malicious misuse by individuals, groups or states?

We do not see any potential for weaponisation of our systems. Specifically, the destruction of living plant matter by either system is essentially impossible because plants have already evolved defenses against the thousands of cellulose degrading organisms which exist; industrial use of cellulases currently depends on extensive pretreatment to remove lignin, a substance extremely difficult to degrade by biological means, and our systems do not bypass this requirement.

Mitigation of risks and BioBrick safety

If your response to any of the questions above is yes:

• Explain how you addressed these issues in project design and while conducting laboratory work.
• Describe and document safety, security, health and/or environmental issues as you submit your parts to the Registry.

In the event that we unexpectedly discover a new safety concern, this page will be updated and any parts on the Registry will be updated with the new information. Any new required procedures in the lab will be implemented. However, we do not anticipate this occurring.

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?

We do not currently have any safety issues to document in the Registry. The BioBricks we have created are not expected to raise any safety issues. However, as mentioned above, if this changes we will take appropriate action.

Oversight

Under what biosafety provisions will / do you operate?

• Does your institution have its own biosafety rules and if so what are they? Provide a link to them online if possible.

Biosafety is covered by [http://www.docs.csg.ed.ac.uk/Safety/Policy/Part6.pdf Part 6] of the university's Health and Safety Policy. Extensive [http://www.ed.ac.uk/schools-departments/health-safety/biosafety/policy/guidance-rules guidance] is also available.

The rules are too extensive to be summarised here, but work must be legal and safe. To ensure this, a risk assessment must be carried out for any work.

• 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, it is a legal requirement in the UK to have a Genetic Modification Safety Committee. Because of the amount of genetic modification going on in the university, each school of the university has [http://www.docs.csg.ed.ac.uk/Safety/bio/GM_safety_committee.pdf at least one] such committee.

The French lab has previously submitted risk assessments for projects involving cellulases, fluorescent proteins, and many other things; since the form of INP we use is not active, it too is covered. So, much of our work is covered by existing risk assessments.

However, we have submitted a new risk assessment form for our project anyway (since some aspects, such as phage work, are not covered by previous risk assessments).

This risk assessment includes information on:

• the host species and strain to be genetically modified (E. coli JM109)
• vectors to be used (pSB1C3 and related vectors, possibly also phagemid vectors)
• the normal actions of the genes inserted (cellulose degradation, ice crystal formation, etc etc)
• transformation procedure (calcium chloride based)
• steps involved in the project (PCR, cloning, assembly)
• potential harmful properties of organisms, genes, vectors, or systems (none in our case)
• the likelihood of the work creating organisms that could survive and flourish in the wild (essentially nil)
• the team members' levels of experience

A copy of the submitted form is available here. This was reviewed by the GM Safety Committee and we received their approval on the 18th of August. No attempt was made to activate the phage system before this time; only preliminary PCR work was done.

• Will / did you receive any biosafety and/or lab training before beginning your project? If so, describe this training.

Our lab workers have three years experience in undergraduate laboratory work and are familiar with basic safety requirements such as lab coats, gloves, etc. We have been instructed in correct disposal procedures for everything we use: physical items generally go to be autoclaved.

• Does your country have national biosafety regulations or guidelines? If so, provide a link to them online if possible.

The relevant laws in the United Kingdom are found in the [http://www.legislation.gov.uk/uksi/2000/2831/contents/made Genetically Modified Organisms (Contained Use) Regulations 2000].

Ideas

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?

Not yet...