We have reproduced our Final Safety Form below. The Buchler lab has a biosafety level of 1, except for the microscopy room (BSL2), which we did not use for this project. We always wore gloves when doing wet-lab work, and did not use any organisms that are classified as dangerous.

Your Training

  1. Have your team members received any safety training yet?
    • Yes, we have already received safety training.
  2. Please briefly describe the topics that you learned about (or will learn about) in your safety training.
    • General laboratory safety, including personal protective equipment, proper handling of biological and hazardous waste, proper handling of sharps, use of fume hoods, and emergency procedures. General chemical safety, including identification and labeling of hazards, proper storage and use of common chemicals, regulations regarding more hazardous substances, waste disposal procedures, and basic first aid and emergency procedures. Fire and life safety, including emergency preparedness and policies.
  3. Please give a link to the laboratory safety training requirements of your institution (college, university, community lab, etc). Or, if you cannot give a link, briefly describe the requirements.
    • Various forms of online and in-person training are required by Duke, depending on the safety levels and hazards you are expected to face. For our purposes, we are required to complete online courses for General Laboratory Safety, General Chemical Safety, and Fire/Life Safety. These courses include online modules and a post-course quiz. A listing of required training is provided here: The training policy is outlined here:

Your Local Rules and Regulations

  1. Who is responsible for biological safety at your institution? (You might have an Institutional Biosafety Committee, an Office of Environmental Health and Safety, a single Biosafety Officer, or some other arrangement.) Have you discussed your project with them? Describe any concerns they raised, and any changes you made in your project based on your discussion.
    • The Duke University and Duke Medicine Occupational and Environmental Safety Office covers safety concerns and compliance. We are working directly within the Buchler and Gersbach labs, which are compliant with and in communication with this office, and we have completed the required safety training that they provide. In addition, all of our work with recombinant DNA has been approved by the Duke Institutional Biosafety Committee.
  2. What are the biosafety guidelines of your institution? Please give a link to these guidelines, or briefly describe them if you cannot give a link.
  3. In your country, what are the regulations that govern biosafety in research laboratories? Please give a link to these regulations, or briefly describe them if you cannot give a link.
    • The OSHA governs biosafety at the national level: North Carolina has its own OSHA-approved state plan for laboratory safety:

Organisms and Parts Used

Species name (including strain) Risk Group Risk Group Source Disease Risk to Humans? Part Number How did you acquire it? How will you use it?
Escherichia coli DH5alpha (K-12) 1 NIH No N/A Buchler Lab via Duke Labs Chassis for cloning and biobrick creation
Escherichia coli DH5alpha-Z1 (K-12) 1 NIH No N/A Dr. Lutz and Expressys Chassis for testing and induction of circuits

Risks of Your Project Now

Please describe risks of working with the biological materials (cells, organisms, DNA, etc.) that you are using in your project. If you are taking any safety precautions (even basic ones, like rubber gloves), that is because your work has some risks, however small. Therefore, please discuss possible risks and what you have done (or might do) to minimize them, instead of simply saying that there are no risks at all.
  1. Risks to the safety and health of team members, or other people working in the lab:
    • The biggest risk we work with is ethidium bromide, which is a mutagen and irritant, although we use it in small concentrations. The E. coli that we use are derived from a K-12 strain and therefore are not particularly dangerous to humans. However, contact with bacteria could still lead to infection or illness, particularly if the bacteria are ingested.
  2. Risks to the safety and health of the general public (if any biological materials escaped from your lab):
    • While our E. coli strains are not specifically a health risk, they could still cause illness and infection if the public is exposed to them. The strains also contain forms of antibiotic resistance, which could make them more dangerous should they escape into the public. We are transforming strains to contain dCas9 derived from S. pyogenes, a bacterium which causes streptococcal skin and throat infection. This gene has no known native pathogenic qualities, and its native endonuclease function is eliminated. In addition, it has been used in bacteria and other cell types without incident. However, there is still a small possibility of unknown functions to this protein that could make bacterial strains more dangerous.
  3. Risks to the environment (from waste disposal, or from materials escaping from your lab):
    • The spread of antibiotic-resistant DNA and cells outside of the laboratory could contribute to the large-scale threat of antibiotic-resistant bacteria in the environment, especially if our plasmids are transferred to more dangerous hosts. Improper disposal of biological materials could allow this to happen in water sources or other public spaces. The same is true for chemicals such as ethidium bromide, which could build up to dangerous quantities in the environment if they are not safely disposed of.
  4. Risks to security through malicious mis-use by individuals, groups, or countries:
    • A malicious user could theoretically take our dCas9-containing strains and use them as one component in the development of a strain that targets human genes or that is resistant to immune system attack. This could potentially produce a bioterrorism agent. However, this would require many further steps of design and the use of other parts not associated with our project, since it is far from the design goals of our circuits.
  5. What measures are you taking to reduce these risks? (For example: safe lab practices, choices of which organisms to use.)
    • Although we use ethidium bromide in low concentrations, we always wear gloves when handling gels as well as any equipment that is used with gels. We have a designated lab area for ethidium bromide and gels, so that other areas are not contaminated. We also wear gloves when handling our DNA or bacteria. In addition, we wipe down surfaces with ethanol before and after handling bacteria, and we bleach any liquid waste before disposal. No food or drink are permitted in the bench area, and we wash our hands when leaving the lab, using the restroom or before eating. Furthermore, the bacteria we are working with are derived from non-pathogenic strains, and therefore do not carry the same level of risk as some other organisms.

Risks of Your Project in the Future

What would happen if all your dreams came true, and your project grew from a small lab study into a commercial/industrial/medical product that was used by many people? We invite you to speculate broadly and discuss possibilities, rather than providing definite answers. Even if the product is "safe", please discuss possible risks and how they could be addressed, rather than simply saying that there are no risks at all.
  1. 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 mis-uses.) Also, what risks might arise if the knowledge you generate or the methodsyou develop became widely available?
    • Our project opens up possibilities for many applications of gene circuits, including targeted gene therapies and industrial bio-production. A bistable gene-targeting switch, if transferred to human cells or viral DNA, could be used to control gene expression in the human body. In fact, the Gersbach lab works on applications of CRISPR technology in these forms. A genetic switch like ours could permit therapies for genetic disorders, cancers, or other challenging diseases. However, a similar system could also be used to target healthy human cells, causing harm either maliciously or accidentally. Even if future versions of our switch do not contain these components, the models and principles that we are developing could help someone else to design a CRISPR switch that targets healthy human cells by controlling the expression of a key gene.
  2. 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.
    • Our project itself is confined to a chassis that is relatively harmless, and our system in its current form is not compatible with human cells or viruses. If the system is ever translated into another more sensitive chassis, further precautions may have to be taken to protect and contain the relevant design information and physical samples. This may include a built-in way to disable the gene circuit, using some harmless inducer molecule, for example. We would also need a plan to prevent others from designing a malicious circuit using our methods and models, possibly by withholding key information on how the circuit is integrated into hosts.

Further comments

Our current project is highly theoretical and experimental, as we are primarily attempting to demonstrate a proof-of-concept approach to ultra-sensitive CRISPR circuits. As we obtain more results regarding the effectiveness of our basic models, we would like to take more time to consider possible applications of the system. This will lead us to have more specific thoughts on the risks of future developments in our project, and any precautions that would need to be taken.