Team:MIT/Safety

• BACKGROUND
• EXPERIMENTS
  Synthetic Promoters microRNA Recombinase Proof of Concept Collaborations
• FUTURE WORK
  Circuit Design Clinical Applications
• HUMAN PRACTICES
  Parts Toolbox Accessible Protocols Integrated Human Practices Public Outreach Interlab Study
• NOTEBOOK
  Lab Notebook Parts Mammalian Parts Collection Attributions Safety
• AWARDS
  HP Silver HP Gold Integrated HP Basic Part
• TEAM

Safety

Laboratory Safety

Before our team began working on our project, we received laboratory safety training in General Biosafety for Researchers, Managing Hazardous Waste, General Chemical Hygiene, and Blood-Borne Pathogens training. These training sessions were given by MIT’s Environmental Health and Safety (EHS) office and prepared us to work with both BL1 materials, such as the E. coli we used for molecular cloning, and BL2 materials, including the human cell lines HEK293, MCF-7, and tHESC, which we used for our experiments. 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. When working with mammalian cells, we worked in biosafety cabinets in a separate room in the lab and wore different lab coats specifically to be used for tissue culture to maximize sterility. When we introduced nucleic acids into mammalian cells, we used either cationic lipid-based transient transfection or electroporation instead of viral infection, since viruses present a greater safety threat. We disposed all chemical reagents, including Qiagen miniprep/midiprep buffers, in clearly labeled containers around the laboratory in accordance with the protocols laid out by their MSDS.

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.