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Engineering Riboswitches in Vivo Using Dual Genetic Selection and Fluorescence-Activated Cell Sorting

Engineering Riboswitches in Vivo Using Dual Genetic Selection and Fluorescence-Activated Cell Sorting

Katharine Page, Jeremy Shaffer, Samuel Lin, Mark Zhang, Jane M Liu

ACS Synth Biol. 2018 Sep 21;7(9):2000-2006.

PMID: 30119599

Abstract:

Riboswitches, noncoding RNAs that bind a small molecule effector to control gene expression at the level of transcription or translation, are uniquely suited to meet challenges in synthetic biology. To expand the limited set of existing riboswitches, we developed a riboswitch discovery platform that couples dual genetic selection and fluorescence-activated cell sorting to identify novel riboswitches from a 108 random-sequence library in which the aptamer domain of the ThiM#2 riboswitch was replaced with an N40 sequence. In a proof-of-principle validation, we identified novel riboswitches for the small molecule theophylline. Our best riboswitch (Hit 3-5) displays 2.3-fold activation of downstream gene expression in the presence of theophylline. Random mutagenesis of Hit 3-5, coupled with selections and screens, afforded improved riboswitches displaying nearly 3-fold activation. To the best of our knowledge, this is the first report of in vivo directed evolution of an aptamer domain to generate a functional riboswitch.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR4243291	Fluorescent Adenosine receptor Antagonist (A-633-AN) synthetic			Price

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