使用5-溴-3-吲哚基-α-L-阿拉伯呋喃糖苷对“无背景”报告基因α-L-阿拉伯呋喃糖苷酶进行原位颜色检测。

In situ color detection of alpha-L-arabinofuranosidase, a "no-background" reporter gene, with 5-bromo-3-indolyl-alpha-L-arabinofuranoside.

作者信息

Berlin W, Sauer B

机构信息

National Institute of Diabetes, Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland 20892-1800, USA.

出版信息

Anal Biochem. 1996 Dec 1;243(1):171-5. doi: 10.1006/abio.1996.0497.

DOI:10.1006/abio.1996.0497

PMID:8954541

Abstract

We describe the synthesis and use of 5-bromo-3-indolyl-alpha-L-arabinofuranoside (5-BI-ara) for the detection of alpha-L-arabinofuranosidase in bacterial colonies. Since the product of 5-BI-ara hydrolysis is an intensely colored indigo precipitate, it is a useful substrate for in situ detection of alpha-L-arabinofuranosidase activity. Here we show that colonies of an Escherichia coli strain expressing a recombinant alpha-L-arabinofuranosidase gene from Streptomyces lividans are readily identified by visual inspection on bacterial plates containing 5-BI-ara. Use of 5-BI-ara should facilitate the detection of endogenous alpha-L-arabinofuranosidase activity in a variety of microorganisms. In addition, since alpha-L-arabinofuranosidase activity is not commonly found in a large number of bacteria, yeast, and animal cells, 5-BI-ara will be useful in exploring the use of genes coding for alpha-L-arabinofuranosidase as gene expression reporters in heterologous systems.

摘要

我们描述了5-溴-3-吲哚基-α-L-阿拉伯呋喃糖苷（5-BI-ara）的合成及其用于检测细菌菌落中α-L-阿拉伯呋喃糖苷酶的方法。由于5-BI-ara水解的产物是一种颜色很深的靛蓝沉淀，它是用于原位检测α-L-阿拉伯呋喃糖苷酶活性的有用底物。在此我们表明，在含有5-BI-ara的细菌平板上通过肉眼观察，很容易鉴定出表达来自淡紫链霉菌的重组α-L-阿拉伯呋喃糖苷酶基因的大肠杆菌菌株的菌落。使用5-BI-ara应有助于检测多种微生物中的内源性α-L-阿拉伯呋喃糖苷酶活性。此外，由于在大量细菌、酵母和动物细胞中通常不存在α-L-阿拉伯呋喃糖苷酶活性，5-BI-ara将有助于探索将编码α-L-阿拉伯呋喃糖苷酶的基因用作异源系统中的基因表达报告基因。