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Author:
Date:
2013-07-20
[Abstract] β-galactosidase and β-glucuronidase enzymes are commonly used as reporters for gene expression from gene promoter-lacZ or uidA fusions (respectively). The protocol described here is a high-throughput alternative to the commonly used Miller assay (Miller, 1972) that utilises a fluorogenic substrate (Fiksdal et al., 1994) and 96-well plate format. The fluorogenic substrates 4-Methylumbelliferyl β-D-galactoside (for β-galactosidase assays) (Ramsay et al., 2013) or 4-Methylumbelliferyl β-D-glucuronide (for β-glucuronidase assays) (Ramsay et al., 2011) are cleaved to produce the fluorescent product 4-methylumbelliferone. Cells are permeabilized by freeze-thawing and lysozyme, and the production of 4-methylumbelliferone is monitored continuously by a ...
[摘要] β-半乳糖苷酶和β-葡糖醛酸糖苷酶通常用作从基因启动子 - lacZ/em或uidA/em融合（分别）的基因表达的报道分子。本文所述的方案是利用荧光底物（Fiksdal等人，1994）和96孔板形式的通常使用的Miller测定法（Miller，1972）的高通量替代物。荧光底物4-甲基伞形基β-D-半乳糖苷（用于β-半乳糖苷酶测定）（Ramsay等人，2013）或4-甲基伞形基β-D-葡萄糖醛酸苷（用于β-葡糖醛酸糖苷酶测定）（ Ramsay等人，2011）切割以产生荧光产物4-甲基伞形酮。通过冷冻 - 融化和溶菌酶使细胞透化，并且通过荧光微板读数器作为动力学测定连续监测4-甲基伞形酮的产生。然后计算荧光增加的速率，从中推断相对基因表达水平。由于高灵敏度基于荧光的4-甲基伞形酮的检测和所收集的高密度时间点，该测定可以在低水平基因表达的定量中提供增加的准确度。该测定需要小的样品体积和最小的制备时间。本方案中概述的透化条件已经针对革兰氏阴性细菌（特别是大肠杆菌和沙雷氏菌）进行了优化，但是可能适合于具有最小优化的其他生物体。
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