Directed Evolution of a Panel of Orthogonal T7 RNA Polymerase Variants for <i>in Vivo</i> or <i>in Vitro</i> Synthetic Circuitry

T7 RNA polymerase is the foundation of synthetic biological circuitry both <i>in vivo</i> and <i>in vitro</i> due to its robust and specific control of transcription from its cognate promoter. Here we present the directed evolution of a panel of orthogonal T7 RNA polymerase:promoter pairs that each specifically recognizes a synthetic promoter. These newly described pairs can be used to independently control up to six circuits in parallel

An <i>in vitro</i> Autogene

Recent technological advances have allowed development of increasingly complex systems for <i>in vitro</i> evolution. Here, we describe an <i>in vitro</i> autogene composed of a self-amplifying T7 RNA polymerase system. Functional autogene templates in cell-free lysate produce T7 RNA polymerase, which amplifies the autogene genetic information through a positive feedback architecture. Compartmentalization of individual templates within a water-in-oil emulsion links genotype and phenotype, allowing evolution

<i>In Vitro</i> Selection for Small-Molecule-Triggered Strand Displacement and Riboswitch Activity

An <i>in vitro</i> selection method for ligand-responsive RNA sensors was developed that exploited strand displacement reactions. The RNA library was based on the thiamine pyrophosphate (TPP) riboswitch, and RNA sequences capable of hybridizing to a target duplex DNA in a TPP regulated manner were identified. After three rounds of selection, RNA molecules that mediated a strand exchange reaction upon TPP binding were enriched. The enriched sequences also showed riboswitch activity. Our results demonstrated that small-molecule-responsive nucleic acid sensors can be selected to control the activity of target nucleic acid circuitry