Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. Paige, J.S., Wu, K.Y. & Jaffrey, S.R. RNA mimics of green fluorescent protein. Science 333, 642–646 (2011).

    Article  CAS  Google Scholar 

  2. Paige, J.S., Nguyen-Duc, T., Song, W. & Jaffrey, S.R. Fluorescence imaging of cellular metabolites with RNA. Science 335, 1194 (2012).

    Article  CAS  Google Scholar 

  3. Song, W., Strack, R.L. & Jaffrey, S.R. Imaging bacterial protein expression using genetically encoded RNA sensors. Nat. Methods 10, 873–875 (2013).

    Article  CAS  Google Scholar 

  4. Strack, R.L. & Jaffrey, S.R. New approaches for sensing metabolites and proteins in live cells using RNA. Curr. Opin. Chem. Biol. 17, 651–655 (2013).

    Article  CAS  Google Scholar 

  5. Ellington, A.D. & Szostak, J.W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).

    Article  CAS  Google Scholar 

  6. Stoltenburg, R., Reinemann, C. & Strehlitz, B. SELEX—a (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol. Eng. 24, 381–403 (2007).

    Article  CAS  Google Scholar 

  7. Zhang, J., Campbell, R.E., Ting, A.Y. & Tsien, R.Y. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol. 3, 906–918 (2002).

    Article  CAS  Google Scholar 

  8. Frommer, W.B., Davidson, M.W. & Campbell, R.E. Genetically encoded biosensors based on engineered fluorescent proteins. Chem. Soc. Rev. 38, 2833–2841 (2009).

    Article  CAS  Google Scholar 

  9. Ibraheem, A. & Campbell, R.E. Designs and applications of fluorescent protein-based biosensors. Curr. Opin. Chem. Biol. 14, 30–36 (2010).

    Article  CAS  Google Scholar 

  10. Kellenberger, C.A., Wilson, S.C., Sales-Lee, J. & Hammond, M.C. RNA-based fluorescent biosensors for live-cell imaging of second messengers cyclic di-GMP and cyclic AMP-GMP. J. Am. Chem. Soc. 135, 4906–4909 (2013).

    Article  CAS  Google Scholar 

  11. Nakayama, S., Luo, Y., Zhou, J., Dayie, T.K. & Sintim, H.O. Nanomolar fluorescent detection of c-di-GMP using a modular aptamer strategy. Chem. Commun. (Camb) 48, 9059–9061 (2012).

    Article  CAS  Google Scholar 

  12. Buckstein, M.H., He, J. & Rubin, H. Characterization of nucleotide pools as a function of physiological state in Escherichia coli. J. Bacteriol. 190, 718–726 (2008).

    Article  CAS  Google Scholar 

  13. Jenison, R.D., Gill, S.C., Pardi, A. & Polisky, B. High-resolution molecular discrimination by RNA. Science 263, 1425–1429 (1994).

    Article  CAS  Google Scholar 

  14. Srinivasan, J. et al. ADP-specific sensors enable universal assay of protein kinase activity. Chem. Biol. 11, 499–508 (2004).

    Article  CAS  Google Scholar 

  15. Ponchon, L. & Dardel, F. Recombinant RNA technology: the tRNA scaffold. Nat. Methods 4, 571–576 (2007).

    Article  CAS  Google Scholar 

  16. Gottschalk, P.G. & Dunn, J.R. The five-parameter logistic: a characterization and comparison with the four-parameter logistic. Anal. Biochem. 343, 54–65 (2005).

    Article  CAS  Google Scholar 

  17. McCombs, J.E. & Palmer, A.E. Measuring calcium dynamics in living cells with genetically encodable calcium indicators. Methods 46, 152–159 (2008).

    Article  CAS  Google Scholar 

  18. Cozzone, A.J. Regulation of acetate metabolism by protein phosphorylation in enteric bacteria. Annu. Rev. Microbiol. 52, 127–164 (1998).

    Article  CAS  Google Scholar 

  19. Lowary, P.T. & Uhlenbeck, O.C. An RNA mutation that increases the affinity of an RNA-protein interaction. Nucleic Acids Res. 15, 10483–10493 (1987).

    Article  CAS  Google Scholar 

  20. Lowry, O.H., Carter, J., Ward, J.B. & Glaser, L. The effect of carbon and nitrogen sources on the level of metabolic intermediates in Escherichia coli. J. Biol. Chem. 246, 6511–6521 (1971).

    CAS  PubMed  Google Scholar 

Download references


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.).

Author information

Authors and Affiliations



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.

Ethics declarations

Competing interests

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

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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).

Download citation

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing