Lac Z gene is a prokaryotic gene that codes for Beta galactosidase enzyme. The e
ID: 220280 • Letter: L
Question
Lac Z gene is a prokaryotic gene that codes for Beta galactosidase enzyme. The enzyme functions by hydrolyzing the ?-galactoside linkage between Galactose and Glucose. Because the enzyme is well understood, and because it uses substrates that produce color upon cleavage, Lac Z is used as a tool in molecular Biology in the following assays:
Blue White selection: You used Lac Z gene in the blue white selection to select the bacterial colonies harboring the recombinant DNA. Colonies that expressed Lac Z produced active Beta galactosidase that turned X-gal substrate into deep blue that you readily saw and distinguished in your plates.
LacZ Reporter Gene: In experiment 8, you used lac Z again. Lac Z has been spliced downstream from the eukaryotic yeast promoter for Galactose metabolism genes (Galactose Regulon). We studied the activity (On/Off, and levels) of the eukaryotic promoter by detecting the protein product of the downstream reporter gene Lac Z. The product of Lac Z , Beta galactosidase enzyme was then used to hydrolyze the ?-galactoside linkage of the substrate ONPG (instead X-Gal) that produces yellow color. The yellow color intensity correlates with the level of expression of lac Z into B-galactosidase that in turn reflects the activity of the eukaryotic promoter in question.
The Question is: Why did we choose to use the substrate ONPG in Experiment 8 instead of X-gal as we did in Lab 5?
Explanation / Answer
ONPG = O-nitrophenyl galactoside
X-gal = 5-bromo-4-chloro-3-indolyl ?-D-galactoside
ONPG is a colorless compound. It is a substrate for beta-galactosidase. When hydrolysis occurs, it produces galactose and o-nitrophenol. O-nitrophenol is a colorimetric substance. So, we can measure the activity of the enzyme/the promoter by measuring the intensity of calorimetric substance produced.
We can also use X-gal. But, hydrolysis of X-gal produces an insoluble blue compound which is not a suitable substrate for calorimetric assays.
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