Lactose Induction of the lac operon in Escherichia coli B23 and its effect on the o-nitrophenyl ß-galactoside Assay

This experiment expanded upon the results of a previous study involving the effect of carbohydrates on the induction of ß-galactosidase, in Escherichia coli. Previous results had indicated that lactose in the growth media caused an apparent decrease in enzyme induction and activity as compared to media containing glycerol. The intent of this experiment was to evaluate whether lactose had a biological effect on the induction of ß-galactosidase. In addition, we investigated whether different methods of lactose addition caused an interference effect with the o-nitrophenyl ß-galactoside (ONPG) enzyme assay through competitive inhibition. The results showed that several factors potentially play a part in the apparent suppression of enzyme activity by lactose using an ONPG assay. The biological effect was seen when lactose was added to the growth media. Enzyme activity increased at a slower rate under this condition than in the glycerol control, but over the length of the experiment, increased to almost the same level. When a large concentration of lactose was added to assay samples, an immediate decrease in enzyme activity was observed, and this suppression continued throughout the experiment. Competition with ONPG limited the effectiveness of the enzyme assay; some of the ß-galactosidase produced by the bacteria probably cleaved lactose instead of the assay indicator. While the ONPG assay is generally a convenient enzyme assay, it was concluded from this study that an alternate type of enzyme assay might be more effective at measuring ß-galactosidase activity in the presence of lactose.