Safety

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

• researcher safety,
• public safety, or
• environmental safety?

Researcher safety

    The majority of our research is conducted with E. coli bacteria. E. coli is a Gram-negative, rod shaped bacterium that is commonly found in the lower intestine of mammals. It is one of the most widely studied prokaryotes, and is used by scientists all over the world as a host organism when working with recombinant DNA, as well as for protein expression.

    The E. coli strains commonly used in the lab, such as DH5α and BL-21 bacteria have been specifically engineered to be grown in a carefully regulated environment that only a laboratory can provide. Due to their sensitivity, they are unable to survive in the human body for an extended period of time. Consequently, the major risk of working with E. coli in the lab is not illness or infection, but rather contamination of other experiments such as eukaryotic cell culture. However, although the risk is deemed acceptable in labs, there is a strict autoclaving policy for all waste fluids, plates, and other waste produced when working with bacteria to mitigate risk to the general public if released by accident. Disposable items, such as tips, agar plates, and spreaders are incinerated, while glassware and other non-disposable equipment is autoclaved or cleaned with 70% isopropanol. Live cultures are treated with bleach using guidelines put forth by The Office of Environmental Health & Radiation Safety (EHRS). Furthermore, all sinks that are used to dispose of liquid waste are treated as potentially hazardous biological waste and are sterilized accordingly by the university. Additionally, these bacteria pose little risk to the environment. These strains of bacteria require conditions that would be unlikely to be found outside of the laboratory.

Public Safety

    The greatest risk E. coli poses to the health and safety of the general public is through intentional, malicious use of the bacteria. While this is always a possibility with any project, our project does present a slightly increased risk due to our use of antibiotic resistance genes as selection factors. This may increase the pathogenicity of the bacteria, but through our strict regulation of the use of these bacteria and stringent disposal guidelines, we believe we have adequately addressed this issue. Furthermore, access to our lab is strictly regulated, and it is extremely difficult for unauthorized individuals to access areas where E. coli is being stored or grown.

Environmental safety

The strains of bacteria that we use in our experiments pose little risk to the environment. These strains of bacteria require conditions that would be unlikely to be found outside of the laboratory.

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?

    While most of our devices and BioBrick parts are pose any safety issues that have not already been addressed, we are planning to biobrick a cytotoxic protein known as clyA. ClyA variants have been used by iGEM teams in the past, however we were unable to determine what precautions they took when using the protein. We also evaluated literature where researchers used clyA, and again, could not find specific safety recommendations. Due to the lack of specific information, we performed all experiments involving clyA under BSL-2 precautions, which was the highest rating available to us. From our experience, we have found that the best resource when dealing with agents of unknown or unclear toxicity, such as clyA is through the department responsible for laboratory safety at whichever institution the research is being performed at. We plan to include the recommendations of our Biosafety Committee and our experiences with clyA in the Registry once our biobrick is submitted.

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?
• If no, which specific biosafety rules or guidelines do you have to consider in your country?

    As one of the world’s foremost research institutions, the University of Pennsylvania has an extensive biological safety manual that we abide by. As a part of these rules, we have submitted a project proposal for EHRS, the equivalent of our Biosafety Committee for review. We are still in the process of working through the process. All members of our lab have completed a basic laboratory safety and biosafety program, as well as further training from postdocs and more experienced lab members. Currently our lab falls under the BSL-1 designation for laboratories, which allows us to perform most of our work. Our mentor Dr. Sarkar has generously provided us with limited BSL-2 lab space that we can use to perform experiments that require more safety precautions. The BSL ratings and other biosafety regulations can be found here: http://www.ehrs.upenn.edu/programs/bio/bsm/principles.html