Safety in the Lab

Our team took precautionary measures to ensure that lab work was being conducted in a safe environment. This was achieved through measures taken prior to entering the lab, during experimental design, and during execution of designed protocols. Prior to beginning lab work, the 2021 team members completed training overseen by the Environmental Health and Safety department at the University of Pittsburgh. The trainings completed covered topics relevant to laboratory safety and discussed laboratory safety during the ongoing pandemic. Relevant topics covered proper laboratory practices such as the use of personal protective gear in the lab, prevention of bloodborne pathogens, social distancing measures within the lab, and proper lab etiquette. While designing protocols, the team opted to use biological and abiotic materials for experiments such that running experimental protocols posed no risk in our laboratories. This involved the use of exclusively non-hazardous reagents in our labs. Additionally, all bacterial strains used in the lab (DH5-alpha, BL21, and K12 JW3933-3 and JW0120 E.coli strains) were classified in risk group one and posed no disease risk to healthy adults. When executing experimental protocols, the team took precautionary measurements to minimize risk of contamination from biological systems involved. Prior to working with bacterial cultures, concentrated alcohol was used alongside flames from Bunsen burners to sterilize the laboratory space. Following sterilization, the use of personal protective equipment, such as face masks, shields, and gloves, was employed to ensure that the team members were not at risk for exposure to biological contaminants or ultraviolet light used in the processing of agarose gels. When leaving the lab, sanitation and proper disposal measures, such as hand washing and the use of biohazard waste containers, were taken. Additional precautions were implemented alongside those taken during lab work in consideration for the ongoing COVID-19 pandemic in the United States. At all points during lab work, the team members wore the proper protective wear and followed precautionary measures advised by the University of Pittsburgh Environmental Health and Safety department.

Safety Outside the Lab

Reagents and biological specimens were only used in experimental protocols inside of the laboratory space. When materials were imported into the lab from providers and manufacturers such as GeneScript, measures were taken to contain packages to avoid contamination of biological material outside of the lab. Protective measurements were taken during all in-person activities conducted by the team members during the course of the project including collaborations, meetings, and project development.

Ethical Considerations

The product which the 2021 team aspires to develop is a tumor diagnostic used in a mammalian system. As such, we will need to discuss with physicians and patient advocates to weigh the ethical implications of using genetically modified organisms, specifically genetically engineered probiotics, as a diagnostic directly introduced to the human digestive tract. Additionally, the biological context in which the probiotic is functioning should be considered as the various parts, such as the fimbrial anchor, involved in probiotic function may pose a risk given a particular set of biological settings if the targeting parts evolve to alter specificity away from the intended CRC tumor signal. This risk can be reduced through extensive testing and application in the lab on cell and mammalian systems. For example, risks involved with fimbriae evolution can be analyzed (ex. through PCR of FimH genes can be used to analyze genetic evolution of the Fimbriae to weigh potential risks.) Additionally, future development of the project must also consider a kill-switch for the engineered bacteria to ensure that colonization longer than the intended period does not take place.