X-gal

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ImageFile = X-Gal.png ImageSize = 200px
IUPACName = 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside
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Section1 = Chembox Identifiers
CASNo = 7240-90-6
PubChem = 65181
SMILES = C1=CC(=C(C2=C1NC=C2O [C@H] 3 [C@@H] ( [C@H] ( [C@H] ( [C@H] (O3)CO)O)O)O)Cl)Br
MeSHName = X-gal
Section2 = Chembox Properties
Formula = C₁₄H₁₅BrClNO₆
MolarMass = 408.629
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Section3 = Chembox Hazards
Solubility =
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X-gal (abbreviated to BCIG) is a galactoside and indole.

Uses

Cloning

In gene cloning, X-gal is used to indicate whether a bacterium expresses the β-galactosidase enzyme, which is encoded by the "lacZ" gene, in a technique called blue/white screening.

X-gal is cleaved by β-galactosidase yielding galactose and 5-bromo-4-chloro-3-hydroxyindole. 5-bromo-4-chloro-3-hydroxyindole then is oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue product. Thus, if X-gal and an inducer of β-galactosidase (usually IPTG) 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 plasmids have acquired foreign genetic material. "E. coli" bacteria which do "not" produce β-galactosidase (coded by lacZ) are transformed 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 "lacZ"α, 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. cite journal
author=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 U S A
volume=97
issue=13
pages=7382–7
year=2000
pmid=10852947
url=http://www.pnas.org/cgi/content/full/97/13/7382
doi=10.1073/pnas.110149297 ] 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 10⁶.

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 [http://www.emdchemicals.com/analytics/literature/Readycult_Consider_the_Differences.pdf] 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.

References