X-gal
Encyclopedia
X-gal is an organic compound
consisting of galactose
linked to a substituted indole
. The compound was synthesized by Jerome Horwitz
and collaborators in Detroit, MI, in 1964. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl galactoside. The X from indoxyl
may well be the source of the X in the X-gal contraction. X-gal is much used in molecular biology
to test for the presence of an enzyme, β-galactosidase
. It is also used to detect activity of this enzyme in histochemistry and bacteriology
. X-gal is one of many indoxyl glycosides and esters that yield insoluble blue compounds similar to indigo
as a result of enzyme-catalyzed hydrolysis.
, and therefore may be hydrolyzed by the β-galactosidase
enzyme which cleaves the β-glycosidic bond
in D-lactose. X-gal, when cleaved by β-galactosidase, yields galactose and 5-bromo-4-chloro-3-hydroxyindole. The latter then spontaneously dimerizes and is oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an intensely blue product which is insoluble. 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. 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.
enzyme in a technique called blue/white screening
. This method of screening is a convenient way of distinguishing a successful cloning product from other unsuccessful ones.
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. 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. 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. However, the multiple cloning site, where a gene of interest may be ligated into the plasmid vector, is located within the lacZα gene. Successful ligation therefore disrupts the lacZα gene, α-complementation is therefore also disrupted and no functional β-galactosidase can form, resulting in white colonies. Cells containing successfully ligated insert can then be easily identified by its white coloration from the unsuccessful blue ones. 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.
.
Organic compound
An organic compound is any member of a large class of gaseous, liquid, or solid chemical compounds whose molecules contain carbon. For historical reasons discussed below, a few types of carbon-containing compounds such as carbides, carbonates, simple oxides of carbon, and cyanides, as well as the...
consisting of galactose
Galactose
Galactose , sometimes abbreviated Gal, is a type of sugar that is less sweet than glucose. It is a C-4 epimer of glucose....
linked to a substituted indole
Indole
Indole 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...
. The compound was synthesized by Jerome Horwitz
Jerome Horwitz
Dr. Jerome P. Horwitz is a globally renowned American scientist; his affiliations included the Barbara Ann Karmanos Cancer Institute, the Wayne State University School of Medicine and the Michigan Cancer Foundation.- Background :Dr...
and collaborators in Detroit, MI, in 1964. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl galactoside. The X from indoxyl
Indoxyl
In chemistry, indoxyl is a nitrogenous substance with the chemical formula: C8H7NO. Indoxyl is isomeric with oxindol and is obtained as an oily liquid....
may well be the source of the X in the X-gal contraction. X-gal is much used in molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
to test for the presence of an enzyme, β-galactosidase
Beta-galactosidase
β-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...
. It is also used to detect activity of this enzyme in histochemistry and bacteriology
Bacteriology
Bacteriology is the study of bacteria. This subdivision of microbiology involves the identification, classification, and characterization of bacterial species...
. X-gal is one of many indoxyl glycosides and esters that yield insoluble blue compounds similar to indigo
Indigo
Indigo is a color named after the purple dye derived from the plant Indigofera tinctoria and related species. The color is placed on the electromagnetic spectrum between about 420 and 450 nm in wavelength, placing it between blue and violet...
as a result of enzyme-catalyzed hydrolysis.
Uses
X-gal is an analog of lactoseLactose
Lactose is a disaccharide sugar that is found most notably in milk and is formed from galactose and glucose. Lactose makes up around 2~8% of milk , although the amount varies among species and individuals. It is extracted from sweet or sour whey. The name comes from or , the Latin word for milk,...
, and therefore may be hydrolyzed by the β-galactosidase
Beta-galactosidase
β-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...
enzyme which cleaves the β-glycosidic bond
Glycosidic bond
In chemistry, a glycosidic bond is a type of covalent bond that joins a carbohydrate molecule to another group, which may or may not be another carbohydrate....
in D-lactose. X-gal, when cleaved by β-galactosidase, yields galactose and 5-bromo-4-chloro-3-hydroxyindole. The latter then spontaneously dimerizes and is oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an intensely blue product which is insoluble. 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. This easy identification of an active enzyme allows the gene for β-galactosidase (the lacZ gene) to be used as a reporter gene
Reporter gene
In molecular biology, a reporter gene is a gene that researchers attach to a regulatory sequence of another gene of interest in cell culture, animals or plants. Certain genes are chosen as reporters because the characteristics they confer on organisms expressing them are easily identified and...
in various applications.
Cloning
In gene cloning, X-gal is used as a visual indication of whether a cell expresses a functional β-galactosidaseBeta-galactosidase
β-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...
enzyme in a technique called blue/white screening
Blue white screen
The blue-white screen is a screening 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...
. This method of screening is a convenient way of distinguishing a successful cloning product from other unsuccessful ones.
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. 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. 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. However, the multiple cloning site, where a gene of interest may be ligated into the plasmid vector, is located within the lacZα gene. Successful ligation therefore disrupts the lacZα gene, α-complementation is therefore also disrupted and no functional β-galactosidase can form, resulting in white colonies. Cells containing successfully ligated insert can then be easily identified by its white coloration from the unsuccessful blue ones. Example of cloning vectors used for this test are pUC19
PUC19
pUC19 is one of a series of plasmid cloning vectors created by Messing and co-workers in the University of California. The p in its name stands for plasmid and UC represents the University in which it was created. It is a circular double stranded DNA and has 2686 base pairs...
, pBluescript
PBluescript
In genetics, pBluescript or pBluescript II is a commercially available phagemid containing several useful sequences for use in cloning with bacteriophage. The sequences include a polylinker sequence , antibiotic resistance sequence to ampicillin and an E. coli and f1 helper phage origin of...
, 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 interactions
In two-hybrid analysis, β-galactosidase may be used as a reporter to identify proteins that interact with each other. In this method, genome libraries may be screened for protein interaction using yeast or bacterial system. Where there is a successful interaction between proteins being screened, it will result to the binding of an activation domain to a promoter. 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. This technique may be limited to screening libraries of size of less than around 106. The successful cleavage of X-gal also creates a noticeably foul odor due to the volatilization of indoleIndole
Indole 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...
.