Safety

Laboratory Safety

Some of the laboratory equipment we used, especially glassware, could potentially become hazardous if it breaks, and several of the reagents we used are skin and/or eye irritant. When working with BL1 materials, such as the E. coli we used for molecular cloning, there is risk of infecting ourselves and contaminating the equipment we use, potentially leading to infection of others. To prepare for this type of work, we received laboratory safety training in General Biosafety for Researchers, Managing Hazardous Waste, and General Chemical Hygiene from MIT’s Environmental Health and Safety (EHS) office. To ensure the safety of ourselves and the biological materials we worked with, we used appropriate personal protective equipment, including gloves, lab coats, and eyewear when necessary. To avoid contamination, we disposed of liquid waste in 10% bleach and cleaned all equipment with 70% ethanol after use. We made sure to discard Qiagen miniprep/midiprep buffers that contain guanidine hydrochloride (which reacts with bleach to produce toxic chlorine gas) in clearly labeled containers around the laboratory.

When working with BL2 materials, including the human cell lines HEK293, MCF-7, and tHESC, which we used for our experiments, there is risk of contracting blood-borne pathogens from these cells. To minimize risk of infection, maintain sterility, and contain the spread of a pathogen in the case that one arises, we worked with all mammalian cells in biosafety cabinets in a separate room in the lab and wore different lab coats from the ones we used when working with bacteria. The use of viruses and viral vectors to introduce nucleic acids into mammalian cells also poses a risk of infecting ourselves and others whom we interact with, so we chose to use cationic lipid-based transient transfection and electroporation instead.

Biological Parts Safety

In our experiments for testing our miRNA sensors, we cotransfected siRNAs into mammalian cells with the sensors. While these siRNAs could be hazardous if they were introduced to human cells, our personal protective equipment was sufficient to protect ourselves from this possibility. Additionally, we never worked with recombinant plasmids encoding miRNAs, and our transfection system was neither infectious nor self-replicating, which lowered the safety risks for working with these materials even lower.

Project Design Safety

In order to greatly improve the safety of our project, we decided to design a diagnostic that works in vitro rather than in vivo, which removes any concern of the project causing any health-related side effects to a patient. Additionally, since our project is a diagnostic, we decided to make the output for a positive diagnosis the production of a yellow fluorescent protein, which is nontoxic to human cells. The only protein we worked with that is toxic to cells at high concentrations is the RNA binding protein L7Ae, which we are using to lower the basal expression of the recombinase TP901. We address this toxicity by expressing the protein under the doxycycline-inducible promoter pTRE, which allows us to externally control the amount of L7Ae being expressed in the cells by growing the cells in an appropriate concentration of doxycycline. The recombinase itself is potentially hazardous, since it performs unidirectional inversion of DNA, but since the plasmid encoding it will be introduced to cells in a biopsy sample rather than a living human, there is no risk of harming any patients with our diagnostic.