BE.109:Protein engineering

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 a natural amino acid in beta-gal with an unnatural amino acid of your choosing, looking at the effect of such a substitution on the expression and activity of the modified enzyme.

Lab handouts
 Day 1: Tools for protein engineering

 Day 2: Assessing beta-galactosidase

 Day 3: Purifying beta-galactosidase

 Day 4: Possible topics for student presentations