Blue-White Selection

Definition

Blue-white screening provides a convenient and powerful way to distinguish bacterial colonies or phage plaques that contain a cloning vector with a DNA insert, from those containing empty vectors with no insert DNA. The method is based on the blue pigment that forms when beta-galactosidase catalyzes hydrolysis of the synthetic substrate X-gal. Hydrolysis of X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactoside) (Horwitz et al. 1964) produces galactose and 5-bromo-4-chloro-3-hydroxyindole (Fig. 1). The latter product then undergoes spontaneous dimerization and oxidation to form a blue-colored indigo pigment (Burstone, 1962). E. coli cells that express beta-galactosidase activity thus form blue colonies when spread on agar plates that contain X-gal, while cells that do not produce active enzyme form white colonies.

Fig. 1

Structure of X-gal and its hydrolysis products