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 acids 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.
Day 1: Tools for protein engineering
Day 2: Assessing beta-galactosidase
Day 3: Purifying beta-galactosidase
Day 4: Possible topics for student presentations
Note: PDF reprints are provided below within the context of fair use. Please obtain copies from the publisher if appropriate.
- A new code for life
EMBO Reports 2004 5(4):336-339
URL Image:Macintosh HD-Users-nkuldell-Desktop-BE109-BE109 lab manual S06-Mod2 (S06)-newcodeforlife EMBORep04.pdf
- The structure of E. coli beta-galactosidase
CR Biol 2005 328(6):549-556
URL Image:Macintosh HD-Users-nkuldell-Desktop-BE109-BE109 lab manual S06-Mod2 (S06)-B-galReview CRBiol05.pdf