How is induced expression of LacZ affected by different pHs?

Abstract

The expression of the lacZ gene was studied to see how different pHs affect its expression. Understanding this will help future lacZ studies in their applications in molecular biology, medicine, and biotechnology. LacZ cloned into the pET (AMP) vector was transformed by heat shock into Sigma Aldrich’s Sig10 chemically competent E. coli cells. Then, Isopropyl-β-D-thiogalactoside (IPTG) was used to induce the expression of lacZ. The gene was then expressed at the following pH values: 3, 4, 7, 10, and 11 to investigate how pH affects the expression of lacZ. Lastly, X-gal, a soluble colorless compound, was hydrolyzed by the beta-galactosidase enzyme to create a blue substrate, and a 10% triton detergent was used to lyse the cells and so that the difference in color could be recorded by a spectrophotometer. Although the goal of this experiment was to produce direct and clear results to indicate a relationship, unfortunately a standard curve wasn’t generated before measuring the optical density of the cultures and therefore the exact molar concentrations of blue from the X-gal cannot be calculated with the data from the spectrophotometry. Additionally, the results of the spectrophotometer seem to indicate that the higher absorbances may not be due to the blue precipitate, as control cultures that did not react with the X-gal also had absorbance values. However, given that the optical densities of each well was approximately the same, the trend that is seen in the absorbance values also cannot be the result of higher pHs killing off cells either.