Blue White Screen - Background

Background

Molecular cloning is one of the most commonly used procedures in molecular biology. A gene of interest may be inserted into a plasmid vector via ligation, and the plasmid is then transformed into Escherichia coli cells. However, not all the plasmids transformed into cells may contain the desired gene insert and checking each individual colony for the presence of the insert is time-consuming, so a method for the detection of the insert is therefore useful for making this procedure less time and labor intensive. One of the early methods developed for the detection of insert is blue-white screening which allows for identification of successful products of cloning reactions through the colour of the bacterial colony.

The method is based on the principle of α-complementation of the β-galactosidase gene. This phenomenon of α-complementation was first demonstrated in work done by Agnes Ullmann in the laboratory of François Jacob and Jacques Monod, where the function of an inactive mutant β-galactosidase with deleted sequence was shown to be rescued by a fragment of β-galactosidase in which that same sequence, the α-donor peptide, is still intact. Langley et al. showed that the mutant non-functional β-galactosidase was lacking in part of its N-terminus with its residues 11—41 deleted, but it may be complemented by a peptide formed of residues 3—90 of β-galactosidase. M13 filamentous phage containing sequence coding for the first 145 amino acid was later constructed by Messing et al., and α-complementation via the use of a vector was demonstrated by the formation of blue plaques when cells containing the inactive protein were infected by the phage and then grown in plates containing X-gal.

The pUC series of plasmid cloning vectors by Vieira and Messing was developed from the M13 system and were the first plasmids constructed to take advantage of this screening method. In this method, DNA ligated into the plasmid disrupts the α peptide and therefore the complementation process, and no functional β-galactosidase can form. Cells transformed with plasmid containing an insert therefore form white colonies, while cells transformed with plasmid without an insert form blue colonies; result of a successful ligation can thus be easily identified by the white coloration of cells formed from the unsuccessful blue ones.