Harnessing the Central Dogma for Stringent Multi-Level Control of Gene Expression

Stringent control of 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. Unfortunately, the ability to precisely regulate gene expression is often hampered by unwanted biomolecular fluctuations and poorly performing genetic parts. In this talk, we show how the central dogma can be harnessed to create what we call Multi-Level Controllers (MLCs) that combine cutting-edge RNA-based regulators with more commonly used protein-based transcription factors (Greco et al. Nature Communications 12, 1738, 2021). 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. Together these features make our MLCs some of the most tightly controlled and high-performance inducible systems built to date. This work provides a major step forward in the tangled world of gene expression control, demonstrating that modern synthetic biology tools can forge a path towards much-needed precise and reliable control for those working with toxic genes and requiring precise regulation and propagation of cellular signal. It also demonstrates the value of exploring more complex and diverse regulatory designs for synthetic biology.

Veronica Greco, Amir Pandi, Tobias J. Erb, Claire S. Grierson, Thomas Gorochowski. Nature Communications. (DOI: 10.1038/s41467-021-21995-7)