Practice question

The lacZ gene encodes β-galactosidase, which hydrolyzes the artificial substrate X-gal into an insoluble blue chromophore. In vectors like pUC19, the multiple cloning site resides within the lacZα fragment encoding the N-terminal α-peptide. Successful ligation of foreign DNA into this site disrupts the open reading frame, preventing formation of active enzyme through insertional inactivation and loss of α-complementation. Without functional β-galactosidase, X-gal remains uncleaved. Consequently, recombinant colonies appear white on indicator plates containing IPTG and X-gal, whereas non-recombinant colonies remain blue.