Harnessing the central dogma for stringent multi-level control of gene expression

Abstract

<jats:title>Abstract</jats:title><jats:p>Strictly controlled inducible gene expression is crucial when engineering biological systems where even tiny amounts of a protein have a large impact on function or host cell viability. In these cases, leaky protein production must be avoided at all costs. Here, we demonstrate how the central dogma offers a simple way to effectively address this challenge. By simultaneously regulating both transcriptional and translational levels, we show how basal expression of an inducible system can be reduced to virtually undetectable levels, with minimal impact on the maximum induced expression rate achieved. Using this approach, we create several stringent expression systems displaying &gt;1000-fold change in their output after induction. Furthermore, we find that multi-level regulation is able to supress transcriptional noise and create a digital-like switch when transitioning between ‘on’ and ‘off’ states. This work provides foundational knowledge and a genetic toolkit of parts to create multi-level gene expression controllers for those working with toxic genes or requiring precise regulation and propagation of cellular signals. It also demonstrates the value of exploring more complex and diverse regulatory designs for synthetic biology.</jats:p>

Highly Reversible Tunable Thermal-repressible Split-T7 RNA polymerases (Thermal-T7RNAPs) for dynamic gene regulation

“…The Thermal-T7RNAPs can be an useful addition for combinatorial multi-level temperature-based gene regulations (e.g. combining transcription, post-transcriptional and translational control) (Greco et al, 2020;Naseri and Koffas, 2020). Potentially, it can be an envisioning tool for controlling the complex inter-population dynamics of microbial consortia in emerging biomedical and bioproduction applications.…”

A standardized genome architecture for bacterial synthetic biology (SEGA)

“…Besides transcription, translation is a major determinant of gene expression, and the ability to manage both provides highly stringent control of synthetic biology designs 43 . To address this, we engineered C2 control elements-translation initiation regions (TIRs)-known to control the speed of translation initiation with a major impact on overall translational efficiency 44,45 .…”

“…Multi-level control of a difficult-to-express gene is enabled by SEGA. The interplay of transcriptional and translational control is critical when it comes to gene expression burden or product toxicity 43 . The standardized setup and the simplistic genome engineering workflow of SEGA with multi-level control might allow for optimizing genomic constructions with targets that show toxic effects.…”

RNA compensation: A positive feedback insulation strategy for RNA-based networks

“…We show that RNA compensation can restore the function of long STAR-based cascades, and eliminate the effects of signal consumption from the steady state behavior of multi-stationary STAR-based circuits. These computational results are broadly relevant to RNA synthetic biology: STARs have been used to create logic gates, cascades and feed-forward loops in combination with many other RNA-based mechanisms, such as small RNA (sRNA) transcriptional repressors, riboregulators, and CRISPR interference (CRISPRi) systems [15,23,22,24]. We anticipate that an effective insulation strategy is necessary to advance towards complex and dynamic RNA circuits.…”