Document Type

Paper- Restricted to Campus Access

Publication Date

4-22-2021

Faculty Mentor

Samantha Wilner

Abstract

The objective of this experiment was to purify and characterize the function of an unknown enzyme through both wet and computational lab protocols. Protein 3H04 was isolated and additional research was conducted using BLAST, PFAM, and DNASU to determine the characteristics and mechanism of this protein. We introduced an expression vector into E. coli via transformation in order to express the protein of interest (POI). Bacteria that took up the expression vector were selected under antibiotic pressure on plates, cultures were inoculated, and then transferred into autoinduction media in order to induce protein expression. The POI was isolated via affinity chromatography, confirmed by SDS PAGE, and the concentration was measured with a Bradford Assay. We successfully isolated 3H04 from E. coli at a concentration of 1.525 mg/mL. Para-nitrophenyl acetate and kinetic assays were also carried out in order to confirm our protein is a hydrolase and determine the Km, Vmax, and Km/Vmax values.

Comments

Presented as part of the Ursinus College Celebration of Student Achievement (CoSA) held April 22, 2021.

The downloadable file is a poster presentation with audio commentary with a run time of 6:00.

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