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Digitalizing heterologous gene expression in Gram-negative bacteria with a portable ON/OFF module

Lookup NU author(s): Dr Angel Goni-Moreno

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Abstract

© 2019 The Authors. Published under the terms of the CC BY 4.0 licenseWhile prokaryotic promoters controlled by signal-responding regulators typically display a range of input/output ratios when exposed to cognate inducers, virtually no naturally occurring cases are known to have an OFF state of zero transcription—as ideally needed for synthetic circuits. To overcome this problem, we have modelled and implemented a simple digitalizer module that completely suppresses the basal level of otherwise strong promoters in such a way that expression in the absence of induction is entirely impeded. The circuit involves the interplay of a translation-inhibitory sRNA with the translational coupling of the gene of interest to a repressor such as LacI. The digitalizer module was validated with the strong inducible promoters Pm (induced by XylS in the presence of benzoate) and PalkB (induced by AlkS/dicyclopropyl ketone) and shown to perform effectively in both Escherichia coli and the soil bacterium Pseudomonas putida. The distinct expression architecture allowed cloning and conditional expression of, e.g. colicin E3, one molecule of which per cell suffices to kill the host bacterium. Revertants that escaped ColE3 killing were not found in hosts devoid of insertion sequences, suggesting that mobile elements are a major source of circuit inactivation in vivo.


Publication metadata

Author(s): Calles B, Goni-Moreno A, de Lorenzo V

Publication type: Article

Publication status: Published

Journal: Molecular Systems Biology

Year: 2019

Volume: 15

Issue: 12

Online publication date: 19/12/2019

Acceptance date: 24/10/2019

Date deposited: 13/01/2020

ISSN (electronic): 1744-4292

Publisher: EMBO Press

URL: https://doi.org/10.15252/msb.20188777

DOI: 10.15252/msb.20188777

PubMed id: 31885200


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