Prior to working in the wet lab, all of our team members completed a Lab Safety for Research Personnel and Hazardous Waste Management online course created by the UCSC Environmental Health and Safety department (EH&S). These courses covered proper personal protective equipment, safe lab practices, lab organization, and proper maintenance and disposal of chemicals and biohazards. After successfully completing and passing these online courses, we received in-lab orientations by our principal investigator, Dr. David Bernick. EH&S supplied our wet lab with traditional white lab coats suitable for BSL1 environments as well as lab safety glasses. Further, we delegated weekly lab chores to maintain a clean lab environment, consistently practiced keeping tidy bench spaces and enforced a policy in which no one was permitted to work in the lab alone. To operate the autoclave in the UC Santa Cruz Baskin Engineering building, we all completed the autoclave safety online course from EH&S and received in-person training by our principal investigator. We were also trained by our Lab Safety Officer, Torrey Brownell, to use the dishwasher in the Baskin Engineering building. The autoclave in our building was often out of order, so we were additionally trained to use autoclaves in two neighboring buildings on campus: the Biomedical and Physical Sciences buildings.
Lauren and Elizabeth practicing safe laboratory habits (proper eye wear, lab coats, gloves, and long hair tied back) while performing an experiment.
In order to validate the functionality of our Ex-4 from both S. cerevisiae and E. coli, a binding affinity assay with its receptor (GLP-1 R) will be performed as opposed to testing our Ex-4 on mammalian cells in our lab. This will allow us to continuously work in a lower risk lab environment in the early stages of our project. Due to the potential hazards associated with mammalian cell cultures, we reached out to the Ku lab at UCSF who are currently culturing mice pancreatic beta cells to monitor insulin production. We plan to have them test our Ex-4 on their mice cell lines and perform an ELISA to further validate our Ex-4.
Having a white list organism was an essential criteria for our team when choosing a host to express our protein. When working with E. coli, special handling took place to prevent any unwanted contamination. E. coli waste was properly disposed of in biohazard waste bins and liquid cultures were discarded accordingly with bleach.
Throughout the course of our project, we met with experts studying S. cerevisiae and protein engineering to ensure we were minimizing any risk that could come from the protocols we are following. As a control mechanism for S. cerevisiae, protein production is dependent on the presence of galactose. S. cerevisiae is commonly known as “baker’s yeast” because it is a GRAS organism [1] found in many baked goods. Due to its non-pathogenic nature, expressing Ex-4 within this organism promotes the safety of both our team members and the intended users of Helo.