X-Gal

X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) is a synthetic substrate used for the detection of lacZ gene activity, which encodes the enzyme β-galactosidase. When X-Gal is hydrolyzed by β-galactosidase, it produces an insoluble blue product, 5,5'-dibromo-4,4'-dichloro-indigo, providing a convenient colorimetric readout for the presence of β-galactosidase activity. This property makes X-Gal widely used in molecular biology, genetics, and microbiology for blue/white screening of recombinant plasmids in Escherichia coli and other bacteria.

Usage in Molecular Biology

In molecular biology, X-Gal is primarily used in the blue/white screening technique. This method allows for the identification of bacterial colonies that have successfully taken up a plasmid vector containing an insert of interest. The plasmid vector typically contains a multiple cloning site (MCS) within the lacZ gene. When a DNA fragment is inserted into the MCS, it disrupts the lacZ gene, preventing it from producing functional β-galactosidase. Bacteria transformed with vectors that have the insert will form white colonies on media containing X-Gal, while those transformed with non-recombinant plasmid (retaining a functional lacZ gene) will produce blue colonies due to the hydrolysis of X-Gal by β-galactosidase.

Chemical Properties

X-Gal is a colorless compound until it is cleaved by β-galactosidase. The resulting product, 5,5'-dibromo-4,4'-dichloro-indigo, is blue and insoluble, allowing for easy visualization. The compound is typically used at a concentration of 40 µg/mL in agar plates, although the concentration can vary depending on the application.

Applications

Beyond blue/white screening, X-Gal is used in other applications that require the detection of β-galactosidase activity. These include:

• Reporter gene assays, where the lacZ gene is fused to a promoter of interest to study gene expression.
• Histology and cytochemistry, for detecting β-galactosidase activity in cells and tissues.
• Genetic engineering for the selection of engineered cells.

Safety and Handling

X-Gal should be handled with care as it is a synthetic compound with potential health risks if not handled properly. It should be stored in a cool, dry place, protected from light to prevent degradation.

See Also

• LacZ
• β-galactosidase
• Plasmid
• Molecular cloning
• Reporter gene