Fowler Casey C, Li Yingfu
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada.
Methods Mol Biol. 2014;1103:177-97. doi: 10.1007/978-1-62703-730-3_14.
A riboswitch is an RNA element that detects the level of a specific metabolite within the cell and regulates the expression of co-transcribed genes. By fusing a riboswitch to a reporter protein in a carefully designed and tested construct, this ability can be exploited to create an intracellular sensor that detects the level of a particular small molecule within live bacterial cells. There is a great deal of flexibility in the design of such a sensor and factors such as the molecule to be detected and the downstream experiments in which the sensor will be applied should guide the specific blueprint of the final construct. The completed sensor plasmid needs to be rigorously tested with appropriate controls to ensure that its dynamic range, signal strength, sensitivity and specificity are suitable for its intended applications. In this chapter, methods for the design, assessment and use of riboswitch sensors are provided along with those for one example application for which riboswitch sensors are ideally suited.
核糖开关是一种RNA元件，可检测细胞内特定代谢物的水平，并调节共转录基因的表达。通过在精心设计和测试的构建体中将核糖开关与报告蛋白融合，可以利用这种能力创建一种细胞内传感器，用于检测活细菌细胞内特定小分子的水平。这种传感器的设计有很大的灵活性，诸如要检测的分子以及将应用该传感器的下游实验等因素应指导最终构建体的具体蓝图。完整的传感器质粒需要用适当的对照进行严格测试，以确保其动态范围、信号强度、灵敏度和特异性适合其预期应用。在本章中，将提供核糖开关传感器的设计、评估和使用方法，以及核糖开关传感器非常适合的一个示例应用的方法。
