X-gal (also abbreviated
BCIG for bromo-chloro-indolyl-galactopyranoside) is an
organic compoundAn organic compound is any member of a large class of chemical compounds whose molecules contain carbon. For historical reasons discussed below, a few types of compounds such as carbonates, simple oxides of carbon and cyanides, as well as the allotropes of carbon, are considered inorganic...
consisting of
galactosideA galactoside is a glycoside containing galactose. The H of the OH group on carbon-1 of galactose is replaced by an organic moiety.Depending on whether the glycosidic bond lies "above" or "below" the plane of the galactose molecule, galactosides are classified as α-galactosides or β-galactosides....
linked to
indoleIndole is an aromatic heterocyclic organic compound. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring. Indole is a popular component of fragrances and the precursor to many pharmaceuticals. Compounds that contain an...
.
Cloning
In gene cloning, X-gal is used to indicate whether a cell expresses the
β-galactosidaseβ-galactosidase, also called beta-gal or β-gal, is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins. Alternate or nicknames are "beta-gal"...
enzyme, which is encoded by the
lacZ geneThe lac operon is an operon required for the transport and metabolism of lactose in Escherichia coli and some other enteric bacteria. It consists of three adjacent structural genes, a promoter, a terminator, and an operator. The lac operon is regulated by several factors including the availability...
, in a technique called
blue/white screeningThe blue-white screen is a molecular technique that allows for the detection of successful ligations in vector-based gene cloning. DNA of interest is ligated into a vector. The vector is then transformed into competent cell . The competent cells are grown in the presence of X-gal...
.
X-gal is cleaved by β-galactosidase yielding galactose and 5-bromo-4-chloro-3-hydroxyindole. The latter is then oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue product. Thus, if X-gal and an inducer of β-galactosidase (usually
IPTGIsopropyl β-
D-1-thiogalactopyranoside, abbreviated IPTG, is a molecular biology reagent.This compound is used as a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon...
) is contained within an agar medium on a culture plate, colonies which have a functional
lacZ gene can easily be distinguished.
This is used when cloning genes as a technique to see whether
plasmidsA cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with the same restriction enzyme, then ligating the fragments together. There are many types...
have acquired foreign genetic material.
E. coli bacteria which do
not produce β-galactosidase (coded by lacZ) are
transformedIn molecular biology, transformation is the genetic alteration of a cell resulting from the uptake, genomic incorporation, and expression of foreign genetic material ....
with a plasmid, some of which contain an insert in the
lacZ open reading frame. For bacteria harboring plasmids with the insert in
lacZ, this gene is disrupted and they are unable to make beta-galactosidase. For bacteria without the insert, β-galactosidase is produced, resulting in a blue colony. This is the case with many commercially available cloning vectors, such as Promega's pGem-T Vectors, which carry
lacZα, a truncated form of β-galactosidase, and require specific
E. coli hosts strains (such as DH5α) to achieve α-complementation.
Reporter
The
lacZ gene may be used as a reporter in combination with growth media containing X-gal. In two-hybrid analysis for example, it is necessary to distinguish between those bacteria in which there is a successful interaction, leading to the binding of an activation domain to a promoter, and those in which there is not. If the promoter is linked to a
lacZ gene, the production of β-galactosidase will be indicated by the production of blue pigment by colonies that host a successful interaction. Due to its manual nature, this technique is limited to situations in which the number of colonies that must be distinguished is less than around 10
6.
Water testing
In addition to use in molecular biology, X-Gal is used to determine
E. coli and coliform content in drinking water samples.
ReadyCult is one such product that is a USEPA approved test for the Presence/Absence of total coliforms and
E. coli in drinking water. This test exploits the fact that naturally occurring
E. coli will have the β-Galactosidase necessary to cleave X-gal and produce an observable blue product. A positive result indicates that the water may be unsafe to drink.