Question

Lab Activity 2: Table 3 CultureRan ulUnused Lysis E. ColiZbuffer Vol. ONPG Conditions | # | 20% | 20% | media for | Reagent | Culture | to bring | (4 mg/mL) Volume to 2 mL for 0.8 mg Lactose Glucose differences in enzyme total LB culture +Lactose Glucose culture + Lactose 2 50 yL 80 yL1020u 1690 u 200 u LB culture +Glucose Lactose culture 4 0 80 10 20 1690 200 BLANK 100 10ul 0ul 0 Lab Activity 2: Table 4 Rxn# Time started Time stopped Elapsed time Aanm Activity 0:00 0:10 0:20 0:28 20:32 20:12 20:22 4:56 1232 1212 1222 0.082 0.193 0.137 0.123 (A420am/time) 0.067 0.159 0.112 0.459 268 L3. In Activity 2, were there any signs that sugar interfered with the assay? Explain.

Answer 1

Escherichia coli have lac operon produces the enzyme β-galactosidase which can break lactose to galactose and glucose. But the gene for β-galactosidase is generally switched off in the absence of Lactose. In this procedure, a sample of E. coli is treated with lactose, and then the β-galactosidase activity of this sample and glucose treated sample are compared. ONPG (ortho-nitrophenyl-β-D-galactoside) is used as a substrate for the enzyme action which produces galactose and a compound which is yellow in alkaline conditions. The intensity (or optical density) of the yellow colour produced is a qualitative indicator or quantitative measure of the β-galactosidase activity.

In Lab activity 2, when we look into the activity of reaction 1 and reaction 3 that is the culture conditions in LB with Lactose and Glucose respectively, it is evident that the Glucose containing LB culture possess more activity when compared to the Lactose containing culture. A similar response is seen when we look into reaction 2 and 4. Thus in the presence of glucose, lactose is least preferred and thus less ONPG is converted to product, that is optical density is low and absorbance is high, and hence the activity is higher when glucose is present in the culture. The procedure indicates that the gene which produces β-galactosidase in E. coli is induced switched on by the presence of lactose. Hence sugars do interfere the assay. Thus the presence of sugar affects the assay reading.