Screening and selection of artificial riboswitches

Methods : a Companion to Methods in Enzymology
Svetlana HarbaughNancy Kelley-Loughnane

Abstract

Synthetic riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules, and a challenge to select this engineered response requires robust screening tools. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer library with a randomized expression platform followed by in vivo selection and screening. In order to determine response to analyte, we developed a dual-color reporter comprising elements of the E. coli fimbriae phase variation system: recombinase FimE controlled by a synthetic riboswitch and an invertible DNA segment (fimS) containing a constitutively active promoter placed between two fluorescent protein genes. Without an analyte, the fluorescent reporter constitutively expressed green fluorescent protein (GFPa1). Addition of the analyte initiated translation of fimE causing unidirectional inversion of the fimS segment and constitutive expression of red fluorescent protein (mKate2). The dual color reporter system can be used to select and to optimize artificial riboswitches in E. coli cells. In this work, the enriched library of aptamers incorporated into the riboswitch architecture reduces the sequence search space by offering a higher p...Continue Reading

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Citations

May 19, 2021·Proceedings of the National Academy of Sciences of the United States of America·Yei-Chen LaiIrene A Chen
Dec 28, 2021·The Journal of Physical Chemistry. B·Andrea Marton Menendez, David J Nesbitt

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