Department of Pharmacology, Weill Medical College, Cornell University, New York, New York, USA.
Nat Protoc. 2014 Jan;9(1):146-55. doi: 10.1038/nprot.2014.001. Epub 2013 Dec 19.
Genetically encoded fluorescent sensors can be valuable tools for studying the abundance and flux of molecules in living cells. We recently developed a novel class of sensors composed of RNAs that can be used to detect diverse small molecules and untagged proteins. These sensors are based on Spinach, an RNA mimic of GFP, and they have successfully been used to image several metabolites and proteins in living bacteria. Here we discuss the generation and optimization of these Spinach-based sensors, which, unlike most currently available genetically encoded reporters, can be readily generated to any target of interest. We also provide a detailed protocol for imaging ADP dynamics in living Escherichia coli after a change from glucose-containing medium to other carbon sources. The entire procedure typically takes ∼4 d including bacteria transformation and image analysis. The majority of this protocol is applicable to sensing other metabolites and proteins in living bacteria.
基因编码荧光传感器可以成为研究活细胞中分子丰度和通量的有价值的工具。我们最近开发了一类新型的由 RNA 组成的传感器，可用于检测各种小分子和未标记的蛋白质。这些传感器基于 Spinach，即 GFP 的 RNA 模拟物，它们已成功用于活体细菌中几种代谢物和蛋白质的成像。在这里，我们讨论了这些基于 Spinach 的传感器的生成和优化，与大多数现有的基因编码报告器不同，它们可以很容易地针对任何感兴趣的目标进行生成。我们还提供了在从含葡萄糖的培养基转变为其他碳源后，对活大肠杆菌中 ADP 动态进行成像的详细方案。整个过程通常需要大约 4 天，包括细菌转化和图像分析。该方案的大部分内容适用于检测活细菌中的其他代谢物和蛋白质。
