Practice question

In blue-white screening, β-galactosidase expressed from plasmid lacZα complements the host ω-fragment to restore active tetrameric enzyme capable of lactose and X-gal metabolism. Under IPTG induction, the functional enzyme hydrolyzes the β-glycosidic bond of the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, X-gal. Cleavage releases 5-bromo-4-chloro-3-hydroxyindole, which spontaneously dimerizes and oxidizes to 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue pigment retained within bacterial colonies. Thus persistent blue color reliably reports intact lacZα activity, while recombinant disruption by insertional inactivation completely prevents color development, enabling clear visual discrimination.