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Using Spinach-based sensors for fluorescence imaging of intracellular metabolites and proteins in living bacteria

Abstract

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.

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Figure 1: Modular strategy for generating Spinach-based sensors.
Figure 2: Characteristics of the ADP sensor.
Figure 3: Live-cell imaging and analysis of endogenous ADP levels.
Figure 4: Background subtraction using the NIS Elements software.
Figure 5: Quantification of mean fluorescence in E. coli.

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Acknowledgements

We thank J.S. Paige and T. Nguyen-Duc for helpful suggestions and Jaffrey lab members for constructive comments on the manuscript. This work was supported by the US National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) grant nos. F32GM106683 (R.L.S.) and R01EB010249 (S.R.J.).

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Contributions

R.L.S., W.S. and S.R.J. conceived the experiments. W.S. had a major role in the development of the Spinach-based sensors. R.L.S. carried out imaging experiments and optimized the imaging protocol. R.L.S. and S.R.J. wrote the manuscript.

Corresponding author

Correspondence to Samie R Jaffrey.

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Competing interests

W.S. and S.R.J. are authors of a US patent application regarding the sensors described in this manuscript.

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Strack, R., Song, W. & Jaffrey, S. Using Spinach-based sensors for fluorescence imaging of intracellular metabolites and proteins in living bacteria. Nat Protoc 9, 146–155 (2014). https://doi.org/10.1038/nprot.2014.001

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