Blue and White selection

Blue or White Selection:

One version of these fusion protein expression vectors places the cloning site at the end of the coding region of the protein β-galactosidase, so that among other things the fusion protein is attached to β-galactosidase and can be recovered by purifying the β-galactosidase activity.

Alterna­tively, placing the cloning site within the β-galactosidase coding region means that cloned inserts disrupt the β-galactosidase amino acid sequence, inactivating its enzymatic activity. This property has been exploited in developing a visual screening protocol that distinguishes those clones in the library that bear inserts from those that lack them.

Cells that have been transformed with a plasmid-based β-galactosidase expression cDNA library (or infected with a similar library constructed in a bacteriophage λ-based β-galactosidase fusion vector) are plated on media containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, or X-gal (Fig. 4.18).

X-gal is a chromogenic substrate, a colourless substance that upon enzymatic reaction yields a coloured product. Following induction with IPTG, bacterial colonies (or plaques) harbouring vec­tors in which the β-galactosidase gene is intact (those vectors lacking inserts) express an active β-galactosi­dase that cleaves X-gal, liberating 5-bromo-4-chloro- indoxyl, which dimerizes to form an indigo blue product.

These blue colonies (or plaques) represent clones that lack inserts. The P-galactosidase gene is inactivated in clones with inserts, so those colonies (or plaques) that remain “white” (actually, colour­less) are recombinant clones.