1. Would any of your project ideas raise safety issues in terms of:
- researcher safety,
- public safety, or
- environmental 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.
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 genes that confer antibiotic resistance 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.
The strains of bacteria that we use in our experiments pose little risk to the environment. This is mainly in part because these strains of bacteria require conditions that would be unlikely to be found outside of the laboratory.
- 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 do not pose any significant 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. We plan to include the recommendations of our Biosafety Committee and our experiences with clyA in the Registry once our biobrick is submitted.
- 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. Through these rules we have been approved for recombinant DNA lab work under a BSL-2 degsignation from the University EHRS after review. 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 full BSL-2 designation for laboratories, which allows us to perform most of our work. Our mentor, Dr. Sarkar, has generously provided us with additional BSL-2 lab space that we can use to perform some of our other experiments. The BSL ratings and other biosafety regulations can be found here
The current safeguards that most research institutions use are designed primarily to protect the researcher from exposure to organisms, DNA, and chemicals that may cause harm. However, there is still room for improvement when considering the control of recombinant DNA (rDNA). For example, while unlikely, there is a possibility that a plasmid, perhaps containing a virulence factor, toxin, or antibiotic resistance gene, could be transferred from a laboratory strain of bacteria to a more hardy strain without the knowledge of the researcher (horizontal gene transfer). Therefore, it may be useful for future iGEM teams to investigate ways that plasmids can be designed to minimize this risk.