X-gal (also abbreviated BCIG for 5-bromo-4-chloro-3-indolyl-β--galactopyranoside) is an
organic compound
In chemistry, organic compounds are generally any chemical compounds that contain carbon-hydrogen or carbon-carbon bonds. Due to carbon's ability to catenate (form chains with other carbon atoms), millions of organic compounds are known. The ...
consisting of
galactose
Galactose (, '' galacto-'' + '' -ose'', "milk sugar"), sometimes abbreviated Gal, is a monosaccharide sugar that is about as sweet as glucose, and about 65% as sweet as sucrose. It is an aldohexose and a C-4 epimer of glucose. A galactose molec ...
linked to a substituted
indole
Indole is an aromatic heterocyclic organic compound with the formula C8 H7 N. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered pyrrole ring. Indole is widely distributed in the natural environmen ...
. The compound was synthesized by
Jerome Horwitz
Jerome Phillip Horwitz (January 16, 1919 – September 6, 2012) was an American scientist; his affiliations included the Barbara Ann Karmanos Cancer Institute, the Wayne State University School of Medicine and the Michigan Cancer Foundation.
...
and collaborators 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
In chemistry, indoxyl is a nitrogenous substance with the chemical formula: C8H7NO. Indoxyl is isomeric with oxindol and is obtained as an oily liquid.
Indoxyl is obtained from indican, which is a glycoside. The hydrolysis of indican yields β ...
may be the source of the X in the X-gal contraction. X-gal is often used in
molecular biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
to test for the presence of an enzyme,
β-galactosidase
β-Galactosidase (EC 3.2.1.23, lactase, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase), is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides.
β ...
, in the place of its usual target, a β-galactoside. It is also used to detect activity of this enzyme in
histochemistry
Immunohistochemistry (IHC) is the most common application of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to ant ...
and
bacteriology Bacteriology is the branch and specialty of biology that studies the morphology, ecology, genetics and biochemistry of bacteria as well as many other aspects related to them. This subdivision of microbiology involves the identification, classificat ...
. X-gal is one of many indoxyl glycosides and esters that yield insoluble blue compounds similar to
indigo dye
Indigo dye is an organic compound with a distinctive blue color. Historically, indigo was a natural dye extracted from the leaves of some plants of the ''Indigofera'' genus, in particular ''Indigofera tinctoria''; dye-bearing ''Indigofera'' pla ...
as a result of enzyme-catalyzed hydrolysis.
Uses
X-gal is an
analog
Analog or analogue may refer to:
Computing and electronics
* Analog signal, in which information is encoded in a continuous variable
** Analog device, an apparatus that operates on analog signals
*** Analog electronics, circuits which use analog ...
of
lactose
Lactose is a disaccharide sugar synthesized by galactose and glucose subunits and has the molecular formula C12H22O11. Lactose makes up around 2–8% of milk (by mass). The name comes from ' (gen. '), the Latin word for milk, plus the suffix '' - ...
, and therefore may be hydrolyzed by the
β-galactosidase
β-Galactosidase (EC 3.2.1.23, lactase, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase), is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides.
β ...
enzyme which cleaves the β-
glycosidic bond
A glycosidic bond or glycosidic linkage is a type of covalent bond that joins a carbohydrate (sugar) molecule to another group, which may or may not be another carbohydrate.
A glycosidic bond is formed between the hemiacetal or hemiketal group ...
in -lactose. X-gal, when cleaved by β-galactosidase, yields galactose and 5-bromo-4-chloro-3-hydroxyindole - 1. The latter then spontaneously dimerizes and is oxidized into 5,5'-dibromo-4,4'-dichloro-
indigo
Indigo is a deep color close to the color wheel blue (a primary color in the RGB color space), as well as to some variants of ultramarine, based on the ancient dye of the same name. The word "indigo" comes from the Latin word ''indicum'', m ...
- 2, an intensely blue product which is insoluble. X-gal itself is colorless, so the presence of blue-colored product may therefore be used as a test for the presence of active β-galactosidase. This also allows for bacterial β-galactosidase (so called ''lacZ '') to be used as a
reporter
A journalist is an individual that collects/gathers information in form of text, audio, or pictures, processes them into a news-worthy form, and disseminates it to the public. The act or process mainly done by the journalist is called journalism ...
in various applications.
Reaction
Cloning
In
gene cloning
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word ''cloning'' refers to the fact that the metho ...
, X-gal is used as a visual indication of whether a cell expresses a functional
β-galactosidase
β-Galactosidase (EC 3.2.1.23, lactase, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase), is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides.
β ...
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
The ''lactose'' operon (''lac'' operon) is an operon required for the transport and metabolism of lactose in ''E. coli'' and many other enteric bacteria. Although glucose is the preferred carbon source for most bacteria, the ''lac'' operon allows ...
gene (lacZα) in the plasmid can complement another mutant
lacZ
The ''lactose'' operon (''lac'' operon) is an operon required for the transport and metabolism of lactose in ''E. coli'' and many other enteric bacteria. Although glucose is the preferred carbon source for most bacteria, the ''lac'' operon allows ...
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 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers. The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the abbreviation for the University of California, where early ...
,
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. Often, the plate containing X-Gal also contain the IPTG (isopropyl β--1-thiogalactopyranoside). IPTG is a chemical structure analogue of lactose. However, IPTG cannot be hydrolyzed by β-galactosidase. IPTG is used as an inducer that binds to lac repressor releasing the DNA and allowing transcription. The presence of IPTG in the agar plate therefore increases the synthesis of β-galactosidase.
[http://www.edvotek.com/300.051205.pdf ]
Variants
X-gal has a number of variants, which are similar molecules with slight differences serving mainly to produce colors other than blue as a signal.
Protein-protein interactions
In
two-hybrid analysis
Two-hybrid screening (originally known as yeast two-hybrid system or Y2H) is a molecular biology technique used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical interactions (such as bindi ...
, 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.
[{{cite journal
, vauthors=Joung J, Ramm E, Pabo C
, title=A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions
, journal=Proc Natl Acad Sci USA
, volume=97
, issue=13
, pages=7382–7
, year=2000
, pmid=10852947
, doi=10.1073/pnas.110149297
, pmc=16554
, bibcode=2000PNAS...97.7382J
, doi-access=free
] 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
Indole is an aromatic heterocyclic organic compound with the formula C8 H7 N. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered pyrrole ring. Indole is widely distributed in the natural environmen ...
.
See also
*
X-Gluc
X-Gluc is a chemical compound with the molecular formula C14H13BrClNO7. It is used as a reagent to detect β-glucuronidase, an enzyme produced by the '' E. coli'' bacterium. It is used to detect ''E. coli'' contamination in food, water and the u ...
References
Galactosides
Indoles
Chloroarenes
Bromoarenes