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Abstract
Previous experiments measuring β -galactosidase ( β -gal) activity using a colourimetric enzyme activity assay have shown that the addition of lactose to cultures induced with isopropylthiogalactoside caused an apparent decrease in induction of the enzyme. This effect stands out as counter-intuitive. We demonstrate that the production of β -gal enzyme protein by E. coli B23 grown in the presence of lactose is in fact higher than that of a glycerol control. This finding was determined quantitatively by measuring the amount of β – gal enzyme protein directly. E. coli B23 was grown in a variety of media, each containing a different carbohydrate, and culture samples were taken over a time course of 60 minutes. Proteins were extracted by bead bashing, and a Bradford assay was used to determine the concentration of protein in each sample. Amounts of β -gal were assessed by a densitometry assay following protein separation and detection by SDS-PAGE and silver staining. Despite low β -gal enzyme activity readings compared to the glycerol control, possibly due to the interference of lactose in the colourimetric assay, the densitometry readings show that this protein was present in significantly larger amounts in the lactose sample compared to the glycerol control. Growth curves for E. coli grown in media containing different carbohydrates were approximately the same, which ensures that differences in enzyme expression at equivalent times were not due to different amounts of bacteria in the cultures. The following experiments show that measuring enzyme activity of β -gal is an inaccurate way of determining β -gal induction when cells are grown in medium containing lactose. Instead, the amount of β -gal protein produced should be measured directly using SDS- PAGE and a suitable protein detection method.