Abstract

Cells use feedback regulation to ensure robust growth despite fluctuating demands for resources and differing environmental conditions. However, the expression of foreign proteins from engineered constructs is an unnatural burden that cells are not adapted for. Here we combined RNA-seq with an in vivo assay to identify the major transcriptional changes that occur in Escherichia coli when inducible synthetic constructs are expressed. We observed that native promoters related to the heat-shock response activated expression rapidly in response to synthetic expression, regardless of the construct. Using these promoters, we built a dCas9-based feedback-regulation system that automatically adjusts the expression of a synthetic construct in response to burden. Cells equipped with this general-use controller maintained their capacity for native gene expression to ensure robust growth and thus outperformed unregulated cells in terms of protein yield in batch production. This engineered feedback is to our knowledge the first example of a universal, burden-based biomolecular control system and is modular, tunable and portable.

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Acknowledgements

This work was supported by EPSRC (grant EP/J021849/1 to F.C., G.-B.S. and T.E.; grant EP/M002306/1 to A.B., O.B. and C.G.; grant EP/P009352/1 to G.-B.S.; Fellowship EP/M002187/1 to G.-B.S.; Fellowship EP/M002306/1 to T.E.), BBSRC/EPSRC SBRC BrisSynBio (grant BB/L01386X/1 to T.E.G.), the Royal Society (Fellowship UF160357 to T.E.G.), the NIHR Imperial Biomedical Research Centre (Y.N.L.) and BBSRC (grant BB/K006290/1 to A.R.A.). F.C. acknowledges the support of the Imperial College London Junior Research Fellowship Scheme. All authors thank G. Cambray for useful discussions.

Author information

Affiliations

  1. Department of Chemical Engineering, Imperial College London, London, UK.

    • Francesca Ceroni
  2. Imperial College Centre for Synthetic Biology, Imperial College London, London, UK.

    • Francesca Ceroni
    • , Alice Boo
    • , Olivier Borkowski
    • , Ali R Awan
    • , Charlie Gilbert
    • , Guy-Bart Stan
    •  & Tom Ellis
  3. Department of Bioengineering, Imperial College London, London, UK.

    • Alice Boo
    • , Olivier Borkowski
    • , Ali R Awan
    • , Charlie Gilbert
    • , Guy-Bart Stan
    •  & Tom Ellis
  4. Department of Medical Biotechnologies, University of Siena, Siena, Italy.

    • Simone Furini
  5. BrisSynBio, University of Bristol, Bristol, UK.

    • Thomas E Gorochowski
  6. ITMAT Data Science Group, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, UK.

    • Yaseen N Ladak

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Contributions

F.C., G.-B.S. and T.E. designed the research; F.C., A.B., C.G. and A.R.A. performed the experiments; F.C., S.F., T.E.G., Y.N.L., G.-B.S. and T.E. analyzed the data; F.C., S.F., T.E.G., O.B., G.-B.S. and T.E. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Guy-Bart Stan or Tom Ellis.

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    Analyzed DH10B RNA-seq data

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    Analyzed MG1655 RNA-seq data

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DOI

https://doi.org/10.1038/nmeth.4635

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