X-gal

X-gal
File:X-Gal.svg
IUPAC name 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside
style="background: #F8EABA; text-align: center;" colspan="2" | Identifiers
CAS number	7240-90-6
PubChem	65181
MeSH	X-gal
SMILES
style="background: #F8EABA; text-align: center;" colspan="2" | Properties
Molecular formula	C14H15BrClNO6
Molar mass	408.629
Except where noted otherwise, data are given for materials in their standard state (at 25 °C, 100 kPa)
Infobox references

X-gal (also abbreviated BCIG for bromo-chloro-indolyl-galactopyranoside) is an organic compound consisting of galactose linked to a substituted indole. It is very heavily used in molecular biology.

Uses

Cloning

In gene cloning, X-gal is used to indicate whether a cell 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. 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 IPTG) is contained within an agar medium on a culture plate, colonies which have a functional lacZ gene can easily be distinguished.

When a technique of cloning plasmid vector genes within bacterial cells is optimal, X-gal is used to visually locate yeast or E. coli colonies that have been transformed by the desired plasmid vector in a blue-white screen. E. coli bacteria, which cannot produce the enzyme β-galactosidase (coded by lacZ gene of the lac operon), are transformed by absorbing the plasmid vectors, which contain an insert in the lacZ open reading frame. Once transformed, the bacteria harboring plasmids are now able to produce the enzyme β-galactosidase which can then cleave the X-gal present within the nutrient agar, resulting in a blue colony. Bacteria colonies that grew from bacteria that were not transformed, cannot cleave X-gal, and thus, remain white. The plasmid vectors can also be coded to disrupt a different bacteria's ability to produce β-galactosidase causing the desired bacteria colonies to grow to be white and non-transformed colonies to grow to be blue. 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.

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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 yeast or 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 10⁶. The successful cleavage of X-gal also creates a noticeably foul odor due to the volatilization of indole.

Water testing

In addition to use in molecular biology, X-Gal is used to determine E. coli and coliform content in drinking water samples.

References

de:5-Brom-4-chlor-3-indoxyl-β-D-galactopyranosid