Festival of Science 2018 was held Friday, April 27, 2018.
Jack Mechler - Biochemistry, Faculty Advisors: Nadia Marano and Lorraine Olendzenski
Isolating and Characterizing Functional Amyloid Fibers from Microbacterium sp.
Functional bacterial amyloids make up an important structural component of biofilms, and little is known about their variance in structure throughout nature. This research aimed to adjust isolation procedures developed by Heather Raimer (2017) for an archaeon, to obtain amyloid fibers from Microbacterium sp. that was isolated from the soil of a pig farm by Abby Korn (2014), and shown to test positive for amyloids using the Thioflavin T (ThT) assay by Hunter Berrus (2015) and Jordan Koloski (2016). ThT assays during different bacterial growth conditions showed that the Microbacterium produce more amyloids closely associated with the cells when grown on agar plates, as opposed to liquid media. Early protein isolation trials showed that amyloids more closely associated with the cells were better candidates for isolation. Therefore, plate-grown amyloids were separated from the cells using sonication, and purified from other components using differential centrifugation and polyacrylamide gel electrophoresis. To further purify this amyloidogenic material, and to characterize the monomers that it is composed of, it was depolymerized in formic acid, further purified with differential centrifugation, dried in a speedvac, and resuspended in both water and formic acid with 1M urea. The sample in formic acid and urea was run on an SDS-page gel to determine the size of its monomers and the extent of the purification, and the sample in water examined under a scanning electron microscope, according to previously derived methods. Isolating and imaging these novel functional amyloids will be another step in our understanding of bacterial amyloids.
Funding: University Fellowship