X-Gal is widely used in molecular biology as chromogenic substrate for β-galactosidase. The enzyme hydrolyses this colourless compound into a colourless intermediate, which then dimerizes and gets oxidized into a blue final product (Figure 1). The difference in colour of X-Gal and its hydrolysed product dimer is crucial for the discrimination between samples containing functional or dysfunctional β-galactosidase.
Figure 1. Schematic representation of X-Gal hydrolysis by β-galactosidase.
For cloning purposes, commercial plasmids (pGEM series) containing lacZ
gene with multiple cloning site (MCS) inserted within, are used in E. coli
for the construction of recombinant vectors. The bacterial cells carrying an empty vector will produce β-galactosidase, which hydrolyses X-Gal and turns the colony blue. The clones carrying vector with a fragment inserted in the MCS will appear white, because the fragment insertion into the lacZ
coding sequence interrupts the synthesis of the enzyme. This approach is used for quick assessment of clones carrying a DNA insert and is often the first step in construction of bespoke recombinant plasmids.
For this application, X-Gal is dissolved in N,N-dimethylformamide (DMF)
and added to agar plates (LB or TY) to final concentration 80 µg/ml. It is used in combination with antibiotic selection marker ampicillin
and inducer of lac operon transcription, IPTG
Yeast two-hybrid assay
This is a method for the study of protein interactions in a heterologous host, Saccharomyces cerevisiae
. The principle is based on the control of the reporter gene lacZ
expression. Commercially available vectors containing DNA binding domain (DBD) and activating domain (AD) of a transcription factor (Gal4) are being fused to the proteins of interest.
Recombinant vectors containing DBD-protein A and AD-protein B gene fusions are co-expressed in yeast. If proteins A and B interact, the DBD and AD domains of the transcription factor localise closely together and trigger lacZ
transcription. In the presence of X-Gal, the production of β-galactosidase causes the colonies to turn blue and allows identification of clones where protein pairs physically interact.