Achieving large dynamic range control of gene expression with a compact RNA transcription-translation regulator

Nucleic Acids Research
Alexandra M Westbrook, Julius B Lucks

Abstract

RNA transcriptional regulators are emerging as versatile components for genetic network construction. However, these regulators suffer from incomplete repression in their OFF state, making their dynamic range less than that of their protein counterparts. This incomplete repression causes expression leak, which impedes the construction of larger synthetic regulatory networks as leak propagation can interfere with desired network function. To address this, we demonstrate how naturally derived antisense RNA-mediated transcriptional regulators can be configured to regulate both transcription and translation in a single compact RNA mechanism that functions in Escherichia coli. Using in vivo gene expression assays, we show that a combination of transcriptional termination and ribosome binding site sequestration increases repression from 85% to 98%, or activation from 10-fold to over 900-fold, in response to cognate antisense RNAs. We also show that orthogonal repressive versions of this mechanism can be created through engineering minimal antisense RNAs. Finally, to demonstrate the utility of this mechanism, we use it to reduce network leak in an RNA-only cascade. We anticipate these regulators will find broad use as synthetic biolog...Continue Reading

References

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Citations

Aug 4, 2018·Wiley Interdisciplinary Reviews. RNA·Radoslaw Pluta, Manuel Espinosa
Jan 23, 2020·Applied Microbiology and Biotechnology·Petra TillAstrid R Mach-Aigner
Jul 10, 2018·Nucleic Acids Research·Daniela PintoGeorg Fritz
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Mar 20, 2019·Journal of Industrial Microbiology & Biotechnology·Waqas AhmedRais Ahmed
Dec 9, 2020·Nucleic Acids Research·Joanne M L HoMatthew R Bennett
Mar 21, 2021·Nature Communications·F Veronica GrecoThomas E Gorochowski
Apr 10, 2021·ACS Synthetic Biology·Cameron J GlasscockJulius B Lucks
Oct 21, 2017·Microbiology Spectrum·Brian P Landry, Jeffrey J Tabor

Methods Mentioned

BETA
biosensor
PCR
FACS
flow cytometry
Fluorescence

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