BE.109:Protein engineering

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BE.109 Laboratory Fundamentals of Biological Engineering

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Module 2

Instructor: Natalie Kuldell

TA: Maria Foley

In this experimental module you will study an enzyme with a remarkable history, beta-galatosidase. "Beta-gal" as it's affectionately called had a starring role in the development of the operon model for gene regulation and continues to be a lab workhorse for gene expression studies. In bacteria, this enzyme hydrolyzes the disaccharide, lactose, into two simpler sugars, glucose and galactose. You will be measuring the efficiency of the enzymatic reaction using an artificial substrate, ONPG, which yields a yellow product when it is cleaved by beta-gal. Using a specialized bacterial strain, you will overexpress beta-gal to purify and analyze. Finally, you will test the effect of replacing the natural leucine amino acids in beta-gal with an unnatural amino acid, 5,5,5-trifluoroleucine, looking at the effect of such a substitution on the expression and activity of the modified enzyme.