Zocca, V. F. B. | Search bioRxiv

bioRxiv2023

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 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 efficientl contained leakage, boosted the expression fold-change from OFF to ON, and decreased the detection limit of the switches by three to four 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. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/536885v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1f62b1borg.highwire.dtl.DTLVardef@882ad5org.highwire.dtl.DTLVardef@1b4138eorg.highwire.dtl.DTLVardef@17001be_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Preventing production escape during scale-up using an engineered glucose-inducible genetic circuit

A proper balance of metabolic pathways is crucial for engineering microbial strains that can efficiently produce biochemicals at 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 of 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. By simulating cultivation scale-up under repressive conditions, we preserved the production capacity of cells, which could be fully accessed by switching to glucose in the final production step. The circuit is also resilient, enabling a quick switch in the metabolic status of the culture. Furthermore, the simulated scale-up process selected best-growing cells without compromising their production capability, leading to higher product formation at the end of the process. As a pathwayindependent circuit activated by the preferred carbon source, our engineered glucose-inducible genetic circuit is broadly useful and imposes not additional cost to traditional production processes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/530450v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@f02f6corg.highwire.dtl.DTLVardef@b9631dorg.highwire.dtl.DTLVardef@11a2309org.highwire.dtl.DTLVardef@f255ac_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗