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From 2014.igem.org

Safety

At the beginning of the summer, each iGEM member received safety training for our lab space at the WPI Life Sciences and Bioengineering Center at Gateway Park in Worcester, Ma. We were trained in safe practices for handling biohazardous and infectious materials including safe working practices, clean up and disposal. We also received training in general laboratory and chemical safety practices, chemical use and disposal, and chemical spill response. Here is a link to WPI’s laboratory safety requirements.

We also ensured to follow the biological safety protocol for our institution at the onset of this project. We discussed our proposed project with David Adams, who handles biological safety at WPI, and he determined our rDNA constructs were within our approved guidelines and did not pose any particular biohazard threats. Here is a link to WPI’s biological safety policies.

When filling out the safety form for iGEM, we were forced to consider not only the immediate risks of our project in the lab, but also the long-term risks possible in the future if our construct was successful. Our responses to the safety form prompts and a picture of one of our lab benches can be seen below.

Shawna and Corbyn's Lab Bench (Inspired by Ginkgo Bioworks).

Immediate Risks

Risks to the safety and health of team members, or other people working in the lab:

The main, though extremely remote, risk involved with working with E. coli K-12 strains is the potential infection and colonization of the gastrointestinal tract. We protect ourselves from this risk by wearing gloves, lab coats and safety glasses at all times while in the lab. We also never eat or drink in the lab, and always wash our hands and wipe the benches down with 70% ethanol before leaving the lab. This website from the EPA summarizes the human health risks of E. coli K-12 strains.

In addition to the E. coli K-12 strain, we are working with a BclA, a protein from B. anthracis, and CAEV p28, a protein from Caprine Arthritis Encephalitis Virus. Because these are single proteins, they should not be harmful to any team members or others working in the lab. Nevertheless, we take the same precautions with BclA and CAEV p28 that we take when handling the E. coli K-12 strain.

Risks to the safety and health of the general public (if any biological materials escaped from your lab):

E coli K-12 survives poorly in external environmental conditions. However if the strain mutated and escaped from the lab, gastrointestinal infection could result. Because antibiotic resistant plasmids are used for cloning in E. coli, the transfer of antibiotic resistant plasmids to human pathogens could also occur. This could result in the formation of a superbug, which could potentially threaten the lives of any people exposed to it.

Risks to the environment (from waste disposal, or from materials escaping from your lab):

As stated above, use of antibiotic resistant plasmids for cloning in E. coli carries the inherent risk of transfer of antibiotic resistance genes to natural environmental bacterial strains. If any clones produced in the lab are not disposed of properly, they could transfer DNA to any naturally occurring bacterium, resulting in the creation of a novel strain that could be potentially harmful for humans or any other organisms that come in contact with it.

Risks to security through malicious misuse by individuals, groups, or countries:

As mentioned above, it is possible that malicious misuse could result in the production of multi-drug resistant pathogenic bacterial strains. Also, it is possible malicious individuals could fuse the BclA N terminal domain to a known human toxin and express it of the cell surface in E. coli, thereby converting the harmless K-12 strain into a pathogen.

What measures are you taking to reduce these risks? (For example: safe lab practices, choices of which organisms to use.)

Our primary defense is good laboratory techniques (use of personal protective equipment, careful lab practices, and good hand hygiene). We bleach all E. coli cultures for 24 hours before disposing of them in the sink, to prevent escape of the cultures from the lab. The only fully functioning organism we are using is the relatively benign K-12 E. coli strain. We chose DNA synthesis, rather than cloning directly from the organisms, for the BclA and CAEV genes so that we could protect ourselves from exposure to these agents.

Long-Term Risks

What new risks might arise from your project's growth? (Consider the categories of risk listed in parts a-d of the previous question: lab workers, the general public, the environment, and malicious misuses.) Also, what risks might arise if the knowledge you generate or the methods you develop became widely available?

Based on these premises, it is likely that our diagnosis system would become a widespread tool for livestock health regulations. Used by both small scale sustenance farmers to corporate scale stockyards, our project would become a cornerstone of the modern livestock health system. As antibacterial resistance concern grows, as well as economic pressure from the funds needed to maintain our current practices, the need for cheap and effective diagnostic tools for livestock will expand exponentially. As part of our current objective, our methods of combating antibiotic resistance in bacteria will be recognized as the winner of the Longitude Prize, allowing us to expand our operations to accommodate this agricultural demand. One of the largest risks associated with this method of diagnosis is mutating pathogenic strains. Our project is currently dependent on very specific antigen-antibody bonds, and does not account for mutagenic strains that may lack the antigen we test for. Another risk is associated with our possible antigen blocking in the future, which involves blocking some our E. coli antigens with synthetic antibodies to better improve agglutination test results. This process has the effect of creating E. coli that may be immune to certain antibodies, and not properly recognized by the immune system.

The two new parts we created for our project pose little safety threat on their own. The BclA is only a membrane localization domain and the CAEV capsid protein does not act as a virus while it is missing its other components. These proteins do not add anything to the behavior or to the biohazardous properties of the chimeric E. coli, merely acting as a target for antibodies. However, if our mechanism were to become widely available, the BclA surface domain could be used to attach malicious proteins to the cell's surface if someone with malicious intentions knew how to carry out the process.

In terms of the agglutination assay, one concern with our project is that native E. coli antigens would produce a false positive result when testing for a particular disease if the host organism has an E. coli infection. While this in itself is not an issue with safety, in order to prevent this false positive, our team discussed the possibility of blocking the native E. coli antigens using either synthetic antibodies or llama/alpaca antibodies, which would leave only the antigen of the disease of interest available for antigen-antibody pairing. In doing this, we would essentially be creating a system for E. coli to prevent itself from being detected or attacked by the host organism's natural immune response. This could lead to an infection that is much more difficult to treat than a normal E. coli infection if a culture affected by the E. coli antigen-binding antibodies were to come into contact with humans or animals.

Does your project currently include any design features to reduce risks? Or, if you did all the future work to make your project grow into a popular product, would you plan to design any new features to minimize risks? (For example: auxotrophic chassis, physical containment, etc.) Such features are not required for an iGEM project, but many teams choose to explore them.

At the current time, our project does not include any inherent safety considerations, so there are no current designed features to reduce risk. In the future, however, if this diagnostic tool were to be utilized large scale, some design features should be implemented in order to minimize potential risk. As mentioned above, in further development of this project, ideally the native E. coli antigens would be blocked using either synthetic antibodies or llama/alpaca antibodies. This would leave only the antigen of the disease of interest available for antigen-antibody pairing on the surface of E. coli. This raises the concern of immune resistant E. coli, and our team considered the possibility of designing a fail-safe that would include a heavy metal inducible promoter, such as arsenic, which would only naturally be present in low concentrations in a host organism. Thus, the synthetic antibody that could block the E. coli’s native antigens would only be produced in vitro. This helps to minimize the risk that our immune resistant bacteria could be weaponized.