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

Cellular reagents for diagnostics and synthetic biology.

We have found that the overproduction of enzymes in bacteria followed by their lyophilization leads to 'cellular reagents' that can be directly used to carry out numerous molecular biology reactions. We demonstrate the use of cellular reagents in a variety of molecular diagnostics, such as TaqMan qPCR with no diminution in sensitivity, and in synthetic biology cornerstones such as the Gibson assembly of DNA fragments, where new plasmids can be constructed solely based on adding cellular reagents. Cellular reagents have significantly reduced complexity and cost of production, storage and implementation, features that should facilitate accessibility and use in resource-poor conditions

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