X-gal

X-gal is a galactoside and indole.

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Uses

Cloning

In gene cloning, X-gal is used to indicate whether a bacterium expresses the beta-galactosidase enzyme, which is encoded by the lacZ gene.

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 are transformed with a plasmid, some of which contain an insert in the lacZ gene. For bacteria harboring plasmids with the insert in the lacZ, this gene is disrupted and they are unable to make beta-galactosidase. For bacteria without the insert, beta 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.

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. ^[1] 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. ^[1]

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® ^[2] 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

1. ^ ^{a b} Joung J, Ramm E, Pabo C (2000). "A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions". Proc Natl Acad Sci U S A 97 (13): 7382-7. PMID 10852947.
2. ^ http://www.emdchemicals.com/analytics/literature/Readycult_Consider_the_Differences.pdf