Signal Amplification for Cell-Free Biosensors, an Analog-to-Digital Converter

Abstract

Toehold switches are biosensors useful for the detection of endogenous and environmental RNAs. They have been successfully engineered to detect virus RNAs in cell-free gene expression reactions. Their inherent sequence programmability makes engineering a fast and predictable process. Despite improvements in the design, toehold switches suffer from leaky translation in the OFF state, which compromises the fold change and sensitivity of the biosensor. To address this, we constructed and tested signal amplification circuits for three toehold switches triggered by Dengue and SARS-CoV-2 RNAs and an artificial RNA. The serine integrase circuit efficiently contained leakage, boosted the expression fold change from OFF to ON, and decreased the detection limit of the switches by 3–4 orders of magnitude. Ultimately, the integrase circuit converted the analog switches’ signals into digital-like output. The circuit is broadly useful for biosensors and eliminates the hard work of designing and testing multiple switches to find the best possible performer.

Development of Int-Plex@ binary memory switch system: plant genome modulation driven by large serine-integrases

“…Our first goal was to understand the behavior of this new binary memory system and ensure that the presence of att sites in tandem would not interfere with expected Int activity. For the functional evaluation, we chose cell-free in vitro transcription- translation reactions (TxTl), given its valuable application as a fast platform for testing Int activity, DNA editing tools and genetic circuits 35,36 . The switch was assembled using TxTl optimized parts, including the degfp gene as a reporter, Ribosome Binding Site (RBS) and the T7Max transcription system 35 (Figure 2A).…”