Dual transcriptional-translational cascade permits cellular level tuneable expression control.

Rosa Morra,Christopher J Robinson,Jason Micklefield,Samantha Halliwell,Sam Hay,Mathew Upton,Neil Dixon,Jayendra Shankar,Lisa Butler

doi:10.1093/nar/gkv912

Abstract

The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems.

Highlights

Most inducible bacterial recombinant expression systems operate at the transcriptional level
We demonstrated that the orthogonal riboswitch can control heterologous gene expression in vivo [20,21]. Using both an inducible promoter and the previously developed orthogonal riboswitch [20], we developed a dual transcriptional-translational ON device to directly control the expression of both T7 bacteriophageRNA polymerase (T7 RNAP), and the recombinant gene of interest in an E. coli host
All cells were grown in TB (2.7% yeast extract, 4.5% glycerol, 1.3% Bactotryptone) or LB medium (0.5% yeast extract, 0.5% NaCl, 1.0% Bactotryptone), supplemented with 0.2% glucose

Summary

Introduction

Most inducible bacterial recombinant expression systems operate at the transcriptional level. RNA polymerase (T7 RNAP) in combination with expression vectors containing the T7 promoter This system is widely used despite the well-studied issues of leaky basal expression and heterogeneous induction response [1]. Inducible microbial recombinant expression systems often display either an all-or-none response with limited titratability, or a heterogeneous response to induction within a host cell population [2,3]. This can be troublesome for the production of technically challenging proteins, where, e.g. aggregation, toxicity and/or solubility are an issue [1].

Methods

Results

Conclusion