Team:Penn/Safety

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 MDS 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 gut for an extended period of time and are engineered to have no known virulence factors present in their genome. 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. 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. Because the genes we are using, Channelrhodopsin2, Aequorin, and Pre-Coelenterazine do not provide these bacteria with any virulence factors nor augmented metabolic capacities, we see no reason for these bacteria to pose a serious risk if released accidentally or purposely. Furthermore, these bacteria pose little risk to the environment, as they are a very common bacterium in the microbiological landscape. 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 mechanisms. 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.

HEK 293T cells are the most commonly used eukaryotic cell line used in labs today. Originally derived from human embryonic kidney cells, they are very easy to transfect and grow. They are used in industry to produce therapeutic proteins, and have been used in research for many years. As with E. coli, there have been a great deal of safety measures built into the HEK293T cells. First and foremost, due to the fact that these cells are eukaryotic cells, they are extremely susceptible to relatively small changes in CO2 concentration, pH, osmolarity, nutrient content, temperature, and attack by pathogens. Due to these vulnerabilities, HEK293T cells are completely incapable of surviving outside of the laboratory environment. They require expensive media and equipment to maintain, and therefore pose little risk to our lab members and the general public, even in the event of accidental release. Furthermore, for many of the same reasons, they also pose minimal risk to the environment. As with E. coli, the major risk arises from intentional misuse. While the cells themselves are not directly dangerous, they can and have been used to grow weaponized viruses (which require a eukaryotic host) which can be exceedingly dangerous. However, this would require a great deal of expertise and time, and due to our strict control of those authorized to work with the cells, we believe that we have minimized the risk our cells pose to the general public and ourselves.

As one of the world’s foremost research institutions, the University of Pennsylvania has an extensive biological safety manual that we abide by. The safety manual can be found here: http://www.cdc.gov/biosafety/ 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 member 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 some of our more dangerous experiments. The BSL ratings and other biosafety regulations can be found here: http://www.cdc.gov/biosafety/