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Tunable protein degradation in bacteria

Nature Biotechnology volume 32, pages 1276–1281 (2014)Cite this article

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Abstract

Tunable control of protein degradation in bacteria would provide a powerful research tool. Here we use components of the Mesoplasma florum transfer-messenger RNA system to create a synthetic degradation system that provides both independent control of steady-state protein level and inducible degradation of targeted proteins in Escherichia coli. We demonstrate application of this system in synthetic circuit development and control of core bacterial processes and antibacterial targets, and we transfer the system to Lactococcus lactis to establish its broad functionality in bacteria. We create a 238-member library of tagged essential proteins in E. coli that can serve as both a research tool to study essential gene function and an applied system for antibiotic discovery. Our synthetic protein degradation system is modular, does not require disruption of host systems and can be transferred to diverse bacteria with minimal modification.

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Figure 1: Protein degradation tag characterization.
Figure 2: Pdt system characterization.
Figure 3: Protease-driven control of a synthetic toggle switch.
Figure 4: Tunable control of endogenous bacterial systems and antibacterial targets.

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Acknowledgements

We are grateful to C. Bashor, M. Lobritz, M. Khalil and D. Dwyer for helpful discussions and critical reviews of the manuscript. This work was supported by funding from the Office of Naval Research (ONR) MURI Program, Defense Threat Reduction Agency grant HDTRA1-14-1-0006 and the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Howard Hughes Medical Institute, Boston University, Boston, Massachusetts, USA

    D Ewen Cameron & James J Collins

  2. Center of Synthetic Biology, Boston University, Boston, Massachusetts, USA

    D Ewen Cameron & James J Collins

  3. Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA

    D Ewen Cameron & James J Collins

  4. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA

    James J Collins

Authors
  1. D Ewen Cameron
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Contributions

D.E.C. and J.J.C. conceived the study, analyzed data and wrote the paper. D.E.C. designed and performed the experiments.

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Correspondence to James J Collins.

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D.E.C. and J.J.C. have submitted an international PCT patent application related to the work described here.

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Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Table 1 (PDF 2125 kb)

Supplementary Table 2

Essential protein degradation library (XLSX 89 kb)

Supplementary Table 3

Primers (XLSX 63 kb)

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Cameron, D., Collins, J. Tunable protein degradation in bacteria. Nat Biotechnol 32, 1276–1281 (2014). https://doi.org/10.1038/nbt.3053

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