X-Gal也稱5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside或5-Bromo-4-chloro-3-indolyl β-D-galactoside，X-Gal是β-半乳糖苷酶(β-galactosidase)的顯色底物，在β-半乳糖苷酶的催化下會產生藍色產物。常用於β-半乳糖苷酶的原位染色檢測以及藍白斑篩選。
基本介紹
- 中文名：Xgal
- 分子式：C14H15BrClNO6
- 分子量：408.63
- CAS Number： 7240-90-6
簡介,英文,
簡介
分子式為C14H15BrClNO6，分子量為408.63，CAS Number 7240-90-6。 X–Gal是β–半乳糖苷酶（β–galactosidase）的底物，水解後呈藍色。基於這個特點，pUC系列載體DNA（或其他帶有lacZ 基因載體DNA）以lacZ 缺失細胞為宿主進行轉化時、或用M13噬菌體載體DNA進行轉染時，如果在平板培養基中加入X–Gal和IPTG，由於β–半乳糖苷酶的α–互補性，可以根據是否呈現白色菌落（或噬菌斑）而方便地挑選出基因重組體。
英文
X-gal 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 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, 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 lacZα, 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. [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.
