General Behavior

  1. x-galX-gal is an analog of lactose, and therefore may be hydrolyzed by the β-galactosidase enzyme which cleaves the β-glycosidic bond in D-lactose.
  2. X-gal, when cleaved by β-galactosidase, yields galactose and 5-bromo-4-chloro-3-hydroxyindole.
  3. X-gal itself is colorless, the presence of blue-colored product therefore may be used as a test for the presence of an active β-galactosidase.
  4. This easy identification of an active enzyme allows the gene for β-galactosidase (the lacZ gene) to be used as a reporter gene in various applications.

Gene Clone Use

  1. In gene cloning, X-gal is used as a visual indication of whether a cell
    expresses a functional β-galactosidase enzyme in a technique called blue/white screening.x-gal chemical structure
  2. This method of screening is a convenient way of distinguishing a successful cloning product from other unsuccessful ones.
  3. The blue/white screening method relies on the principle of α-complementation of the β-galactosidase gene, where a fragment of the lacZ gene (lacZα) in the plasmid can complement another mutant lacZ gene (lacZΔM15) in the cell.
  4. Both genes by themselves produce non-functional peptides, however, when expressed together, as when a plasmid containing lacZα is transformed into a lacZΔM15 cells, they form a functional β-galactosidase.
  5. The presence of an active β-galactosidase may be detected when cells are grown in plates containing X-gal, the blue-colored product precipitated within cells resulted in the characteristic blue colonies.
  6. However, the multiple cloning site, where a gene of interest may be ligated into the plasmid vector, is located within the lacZα gene.
  7. Successful ligation therefore disrupts the lacZα gene, α-complementation is therefore also disrupted and no functional β-galactosidase can form, resulting in white colonies.
  8. Cells containing successfully ligated insert can then be easily identified by its white coloration from the unsuccessful blue ones.
  9. Example of cloning vectors used for this test are pUC19, pBluescript, pGem-T Vectors, and it also requires the use of specific E. coli host strains such as DH5α which carries the mutant lacZΔM15 gene.

Protein-Protein Interaction

  1. In two-hybrid analysis, β-galactosidase may be used as a reporter to identify proteins that interact with each other.
  2. In this method, genome libraries may be screened for protein interaction using yeast or bacterial system.
  3. Where there is a successful interaction between proteins being screened, it will result to the binding of an activation domain to a promoter.
  4. If the promoter is linked to a lacZ gene, the production of β-galactosidase, which results in the formation of blue-pigmented colonies in the presence of X-gal, will therefore indicate a successful interaction between proteins.
  5. This technique may be limited to screening libraries of size of less than around 10.
  6. The successful cleavage of X-gal also creates a noticeably foul odor due to the volatilization of indole.

 

Sources: Horwitz JP and 7 others, 1964. Kiernan JA 2007. Joung J, Ramm E, Pabo C (2000).

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    X-Gal
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