Document Type

Paper- Restricted to Campus Access

Publication Date

4-22-2021

Faculty Mentor

Samantha Wilner

Abstract

The sequence and structure of 4DIU protein have been identified, but its function remains unknown. Thus, this research was conducted to elucidate the function of 4DIU. BLAST and Pfam computational results suggest that 4DIU belongs to the alpha beta hydrolase superfamily and contains an aminopeptidase domain. We hypothesize that 4DIU is a carboxylesterase based on sequence alignments using BLAST. In lab, E. coli was transformed with a pET21_NESG expression vector containing the 4DIU gene and an ampicillin resistance gene to select for transformed colonies. Colonies were grown on media containing ampicillin to select for the transformed bacteria. The transformants were then grown in autoinduction media to amplify 4DIU expression and were subsequently lysed using a freeze-thaw protocol in order to extract the amplified protein. Next, 4DIU was purified via nickel-agarose affinity chromatography, and the elution fractions were analyzed using SDS-PAGE. The gel revealed a large, single band corresponding to the 28.5 kDa molecular weight of 4DIU protein. A protein concentration of 3.78 mg/mL was identified via a Bradford assay. Kinetics assays involving p-nitrophenyl acetate as a substrate were used to analyze the activity and potential hydrolytic function of 4DIU protein.

Comments

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

The downloadable .mp4 video file is a poster presentation with audio commentary with a run time of 6:17.

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