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

Abstract

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, but ideally without affecting the achievable range of expression. Here, we demonstrate how the central dogma offers a simple way to effectively address this challenge. By simultaneously regulating both transcription and translation, we show how relative basal expression of an inducible system can be greatly reduced, with minimal impact on the maximum induced expression rate. Using this approach, we create several stringent expression systems displaying >1000-fold change in their output after induction in vivo and up to a 350-fold change when used in a cell-free expression system. Furthermore, we find that multi-level regulation is able to suppress transcriptional noise and creates 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.