Blue White Screen - Molecular Mechanism

Molecular Mechanism

β-galactosidase is a protein encoded by the lacZ gene of the lac operon, and it exists as a homotetramer in its active state. However, a mutant β-galactosidase derived from the M15 strain of E. coli has its N-terminal residues 11—41 deleted and this mutant, the ω-peptide, is unable to form a tetramer and is inactive. This mutant form of protein however may return fully to its active tetrameric state in the presence of an N-terminal fragment of the protein, the α-peptide. The rescue of function of the mutant β-galactosidase by the α-peptide is called α-complementation.

In this method of screening, the host E. coli strain carries the lacZ deletion mutant (lacZΔM15} which contains the ω-peptide, while the plasmids used carry the lacZα sequence which encodes the first 59 residues of β-galactosidase, the α-peptide. Neither are functional by themselves. However, when the two peptides are expressed together, as when a plasmid containing the lacZα sequence is transformed into a lacZΔM15 cells, they form a functional β-galactosidase enzyme.

The blue/white screening method works by disrupting this α-complementation process. The plasmid carries within the lacZα sequence an internal multiple cloning site (MCS). This MCS within the lacZα sequence can be cut by restriction enzymes so that the foreign DNA may be inserted within the lacZα gene, thereby disrupting the gene and thus production of α-peptide. Consequently, in cells containing the plasmid with an insert, no functional β-galactosidase may be formed.

The presence of an active β-galactosidase can be detected by X-gal, a colourless analog of lactose that may be cleaved by β-galactosidase to form 5-bromo-4-chloro-indoxyl, which then spontaneously dimerizes and oxidizes to form a bright blue insoluble pigment 5,5'-dibromo-4,4'-dichloro-indigo. This results in a characteristic blue colour in cells containing a functional β-galactosidase. Blue colonies therefore show that they may contain a vector with an uninterrupted lacZα (therefore no insert), while white colonies, where X-gal is not hydrolyzed, indicate the presence of an insert in lacZα which disrupts the formation of an active β-galactosidase.