X-gal is cleaved by β-galactosidase yielding galactose and 5-bromo-4-chloro-3-hydroxyindole. The latter is then oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue product. Thus, if X-gal and an inducer of β-galactosidase (usually IPTG) is contained within an agar medium on a culture plate, colonies which have a functional lacZ gene can easily be distinguished.
When the technique of cloning plasmid vector genes within bacterial cells is optimal, X-gal is used to visually locate yeast or E. coli colonies that have been transformed by the desired plasmid vector in a blue-white screen. E. coli bacteria, which cannot produce the enzyme β-galactosidase (coded by lacZ gene of the lac operon), are transformed by absorbing the plasmid vectors, which contain an insert, in the lacZ open reading frame. After the transformation process, the bacteria is spread on nutrient agar plates, which mostly contain antibiotics as well. Most commercially available vectors contain an antibiotic-resistant gene. Successfully transformed bacteria has a truncated β-galactosidase gene, causing white colonies on the plate. Bacteria transformed by empty vectors, which do not contain an insert in the lacZ open reading frame, are now able to produce the enzyme β-galactosidase which can then cleave the X-gal present within the nutrient agar, resulting in a blue colony. Bacteria colonies that grew from bacteria that were not transformed do not contain this antibiotic-resistance, and thus, die. The plasmid vectors can also be coded to disrupt a different bacteria's ability to produce β-galactosidase, causing the desired bacteria colonies to grow to be white and non-transformed colonies to grow to be blue. This is the case with many commercially available cloning vectors, such as Promega's pGem-T Vectors, which carry lacZα, a truncated form of β-galactosidase, and require specific E. coli hosts strains (such as DH5α) to achieve α-complementation.
Category:Galactosides Category:Indoles Category:Organochlorides Category:Organobromides
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