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Systematic characterization of maturation time of fluorescent proteins in living cells

Abstract

The slow maturation time of fluorescent proteins (FPs) limits the temporal accuracy of measurements of rapid processes such as gene expression dynamics and effectively reduces fluorescence signal in growing cells. We used high-precision time-lapse microscopy to characterize the maturation kinetics of 50 FPs that span the visible spectrum at two different temperatures in Escherichia coli cells. We identified fast-maturing FPs from this set that yielded the highest signal-to-noise ratio and temporal resolution in individual growing cells.

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Figure 1: Maturation kinetics and their impact on fluorescence signal in growing cells.
Figure 2: Impact of maturation time on transcription dynamics.

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Acknowledgements

Thanks to A. Mendoza-García for help with spectroscopy, V. Pham for help with SDS-PAGE, P.J. Choi for the tsr-venus sequence, and J. Paulsson for useful comments. This work was partially supported by a fellowship from CONACYT-México and from the PEW Latin American Fellows Program to E.B.; a, NSF GRFP fellowship and Harvard University Ashford Fellowship to J.M.K. This work was supported by NSF 1615487, 1410176, DARPA HR0011-16-2-0049, and NIH 1R21AI094363.

Author information

Authors and Affiliations

Authors

Contributions

E.B. and P.C. conceived the project and analyzed the data. E.B. performed cloning, biochemistry, single-cell experiments and software design for single-cell analysis. J.M.K. assisted with cloning and single-cell experiments. E.B. and J.M.K. designed software for time-lapse microscopy. E.B. and P.C. wrote the manuscript with critical revisions by J.M.K. P.C. supervised the project.

Corresponding author

Correspondence to Philippe Cluzel.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–26, Supplementary Tables 1 and 2, and Supplementary Note (PDF 10330 kb)

Life Sciences Reporting Summary

Supplementary Protocol

Measurement of maturation kinetics in living Escherichia coli cells

Supplementary Data 1

Mean single-cell fluorescence curves. Mean single-cell fluorescence curves used to calculate maturation times in Table 1.

Supplementary Data 2

Gel densitometry calculations. SDS-PAGE densitometry calculations used to estimate relative FP expression level in E. coli.

Supplementary Data 3

SDS-PAGE images. Gel images used to quantify the relative FP expression level in E. coli.

Supplementary Data 4

Fluorescence signal in growing cells measured using flow cytometry. Fluorescence mean and variance of growing cell cultures measured using flow cytometry.

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Balleza, E., Kim, J. & Cluzel, P. Systematic characterization of maturation time of fluorescent proteins in living cells. Nat Methods 15, 47–51 (2018). https://doi.org/10.1038/nmeth.4509

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