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, but without affecting the achievable range of expression. Here, we demonstrate how the central dogma offers a simple solution to this challenge. By simultaneously regulating transcription and translation, we show how basal expression of an inducible system can be reduced, with little impact on the maximum expression rate. Using this approach, we create several stringent expression systems displaying >1000-fold change in their output after induction and show how multi-level regulation can suppress transcriptional noise and create digital-like switches between ‘on’ and ‘off’ states. These tools will aid those working with toxic genes or requiring precise regulation and propagation of cellular signals, plus illustrate the value of more diverse regulatory designs for synthetic biology.

Context-Dependent Stability and Robustness of Genetic Toggle Switches with Leaky Promoters

“…Bioenergetic cost associated with the existence and expression of a gene is not a novel concept [77]. Recent advances in experimental techniques enable not only the precise characterization and subdivision of this burden [61,78,79], but also its accurate and multilevel control [80]. Complementing these high-throughput technologies, computational tools and quantitative models enable the design of complex biosystems [3,4,30,81].…”

Strategies for Improving Small-Molecule Biosensors in Bacteria

“…This work introduced conditionally silent leucine-to-amber mutations into reporter genes to make their correct translation dependent upon supP expression, and notably used a toxic mutagenic reporter system to demonstrate that this approach can achieve undetectable levels of leaky expression in the absence of an inducer [ 174 ]. In another study, Greco et al used a toehold switch (THS) translational regulator as well as a small transcription activating RNA (STAR) as leak dampeners to improve IPTG detection using lacI [ 175 ]. Expression of the THS and STAR elements was linked to correct translation initiation and transcription initiation, respectively, of the reporter gene, with the structured RNA elements preventing these processes from occurring in the absence of ligand [ 175 ].…”

“…In another study, Greco et al used a toehold switch (THS) translational regulator as well as a small transcription activating RNA (STAR) as leak dampeners to improve IPTG detection using lacI [ 175 ]. Expression of the THS and STAR elements was linked to correct translation initiation and transcription initiation, respectively, of the reporter gene, with the structured RNA elements preventing these processes from occurring in the absence of ligand [ 175 ]. Another strategy detailed by Fernandez-Rodriguez et al uses multiple plant proteases as leak dampeners, improving the output of an AND gate built from the biosensors tetR and lacI [ 176 ].…”

Towards an engineering theory of evolution

“…Similarly, proteins that are more thermodynamically stable may also be more evolvable 63 . Systems could be buffered against environmental and genetic perturbations through the use of a negative feedback [64][65][66] , tunable genetic parts 67 , stringent multi-level regulation 68 or the application of other control engineering principles 69 .…”