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A transcription activator–like effector (TALE) induction system mediated by proteolysis

Nature Chemical Biology volume 12, pages 254–260 (2016)Cite this article

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Abstract

Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator−like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications owing to their customizable DNA-binding specificity. In this work we expanded the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded after induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator-agnostic.

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Figure 1: Schematic of the TALE-TEV repression-induction system.
Figure 2: Characterization of the proof-of-concept TALE-TEV system.
Figure 3: Comparison of the TALE-TEV system to the LacI-IPTG system.
Figure 4: Characterization of TALE scaffolds with alternative TEV protease cleavage sites.
Figure 5: Targeting chromosomal loci and multiplexing using the TALE-TEV system.
Figure 6: Derepression by sequestration of the TALE.

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Acknowledgements

This work was funded by the US National Science Foundation (NSF) (CBET-114678) and The William F. Vilas Trust. M.F.C. and M.C.P. were supported by NHGRI Genomic Sciences Training Program (T32 HG002760). A.L.M. was supported by NSF SEES fellowship (GEO-1215871). M.C.P. was supported by a US National Institutes of Health (NIH) Biotechnology Training Program (T32 GM008349). We thank D. Waugh for providing guidance on TEV protease variants, and J. Cameron for assistance with taking the photographs that appear in this paper.

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Author notes

  1. Matthew F Copeland & Charles B Johnson

    Present address: Present addresses: The Procter & Gamble Co., Mason, Ohio, USA (M.F.C.); Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, St. Louis, Missouri, USA (C.B.J.).,

  2. Matthew F Copeland and Mark C Politz: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA

    Matthew F Copeland, Mark C Politz, Charles B Johnson, Andrew L Markley & Brian F Pfleger

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Contributions

M.F.C. and B.F.P. conceived the study. M.F.C. and M.C.P. designed and performed the experiments with the following exceptions. A.L.M. proposed, and helped design and perform, the TEV Km mutant experiment. C.B.J. assisted with preliminary experimental work. M.F.C. and M.C.P. analyzed the data. M.F.C., M.C.P. and B.F.P. wrote the manuscript.

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Correspondence to Brian F Pfleger.

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Copeland, M., Politz, M., Johnson, C. et al. A transcription activator–like effector (TALE) induction system mediated by proteolysis. Nat Chem Biol 12, 254–260 (2016). https://doi.org/10.1038/nchembio.2021

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