Pratte Brenda S, Thiel Teresa
Department of Biology, University of Missouri - St. Louis, St. Louis, MO, USA.
Bio Protoc. 2017 Jan 5;7(1):e2084. doi: 10.21769/BioProtoc.2084.
One of the most successful fluorescent proteins, used as a reporter of gene expression in many bacterial, plant and animals, is green fluorescent protein and its modified forms, which also function well in cyanobacteria. However, these fluorescent proteins do not allow rapid and economical quantitation of the reporter gene product, as does the popular reporter gene , encoding the enzyme β-galactosidase. We provide here a protocol for the localization of β-galactosidase activity in cyanobacterial cells. This allows the same strain to be used for both a simple, quantitative, colorimetric assay with the substrate ortho-nitrophenyl-β-galactoside ( and for sensitive, fluorescence-based, cell-type localization of gene expression using 5-dodecanolyaminofluorescein di-β-D-galactopyranoside (C12-FDG).
最成功的荧光蛋白之一是绿色荧光蛋白及其修饰形式，它在许多细菌、植物和动物中用作基因表达的报告基因，在蓝细菌中也能很好地发挥作用。然而，这些荧光蛋白不像常用的编码β-半乳糖苷酶的报告基因那样，能够对报告基因产物进行快速且经济的定量分析。我们在此提供一种在蓝细菌细胞中定位β-半乳糖苷酶活性的方案。这使得同一菌株既能用于与底物邻硝基苯基-β-半乳糖苷进行简单的定量比色测定，又能用于使用5-十二烷氨基荧光素二-β-D-吡喃半乳糖苷（C12-FDG）对基因表达进行基于荧光的敏感细胞类型定位。
