Laboratory Exercise To Measure Restriction Enzyme Kinetics

Abstract

Enzymes are ubiquitous in the fields of biology and microbiology, catalyzing critical reactions and enabling a broad range of biotechnological applications. Despite the important role that enzyme catalysis plays in biological processes, undergraduate students often struggle to understand enzyme kinetics in the classroom. In an attempt to improve students’ understanding of the topic, we present a relatively short and inexpensive laboratory activity designed to give students hands-on experience with generating and manipulating enzyme kinetic data. Students perform restriction digests of DNA at various time points, visualize the reaction products on an agarose gel, and quantify their data in order to construct Lineweaver-Burk plots which compare the effects of a restriction enzyme and its engineered version. The activity may be completed in a single two-hour lab session and, unlike other enzyme assays designed for laboratory courses, does not require a microplate reader to complete. The activity allows students to see connections between a visual data set and quantitative kinetic data, in order to solidify their understanding of enzyme kinetics. Students also learn the skills of gel electrophoresis and image quantification using ImageJ software. This lab activity is ideal for undergraduate laboratory courses which address enzyme kinetics and DNA technology.