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Riboswitches are cis-acting RNA devices in mRNAs that control gene expression in response to chemical inputs. As RNA aptamers that recognize diverse classes of molecules can be isolated by in vitro selection, synthetic riboswitches hold promise for various applications in synthetic biology. One of the major drawbacks of riboswitches, however, is their limited dynamic range. A high level of gene expression in the OFF state (leakage) is also a common problem. To address these challenges, we designed and constructed a dual-riboswitch plasmid in which two genes are controlled by theophylline-activated riboswitches. One riboswitch controls the gene of interest, and another riboswitch controls RepL, a phage-derived replication protein that regulates the plasmid copy number. This single-plasmid system afforded an ON/OFF ratio as high as 3900. Furthermore, we used the system to control CRISPR interference (CRISPRi) targeting endogenous genes, and successfully observed expected phenotypic changes in Escherichia coli.

Riboswitch Signal Amplification by Controlling Plasmid Copy Number