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

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.

Preventing Production Escape Using an Engineered Glucose-Inducible Genetic Circuit.

A proper balance of metabolic pathways is crucial for engineering microbial strains that can efficiently produce biochemicals on an industrial scale while maintaining cell fitness. High production loads can negatively impact cell fitness and hinder industrial-scale production. To address this, fine-tuning gene expression using engineered promoters and genetic circuits can promote control over multiple targets in pathways and reduce the burden. We took advantage of the robust carbon catabolite repression system of Bacillus subtilis to engineer a glucose-inducible genetic circuit that supports growth and production. The circuit is resilient, enabling a quick switch in the production status when exposed to the correct carbon source. By performing serial cultivations for 61 generations under repressive conditions, we preserved the production capacity of the cells, which could be fully accessed by switching to glucose in the next cultivation step. Switching to glucose after 61 generations resulted in 34-fold activation and generated 70% higher production in comparison to standard cultivation in glucose. Conversely, serial cultivation under permanent induction resulted in 62% production loss after 67 generations alongside an increase in the culture growth rate. As a pathway-independent circuit activated by the preferred carbon source, our engineered glucose-inducible genetic circuit is broadly useful and imposes no additional cost to traditional production processes.

Expanding the Functionality of an Autoinduction Device for Repression of Gene Expression in Bacillus subtilis.

Autonomous control of gene expression through engineered quorum-sensing processes is broadly applicable to biosynthetic pathways, including simultaneous control of different genes. It is also a powerful tool for balancing growth and production. We had previously engineered a modular autoinduction device for the control of gene expression in B. subtilis. Now, we expand its functionality to repress gene expression autonomously. The engineered R8 promoter responds to AHL accumulation in the culture medium. In a riboflavin-producing strain, the AHL-Lux complex exerts 5-fold repression on the R8-driven expression of the flavokinase/FAD synthetase gene ribC, resulting in a higher titer of the vitamin. We engineered a strain able to autonomously induce and repress different genes simultaneously, demonstrating the potential of the device for use in metabolic engineering.