Construction of a Modular Framework for Orthogonal and Predictable Gene Expression in Prokaryotes

Synthetic gene circuits can produce complex biomolecules and implement complex computations, but their performance is often limited to model organisms. By incorporating orthogonal transcriptional control elements in the design and construction of synthetic circuits it should prove possible to increase robustness, predictability, and portability across diverse organisms, while mitigating potential crosstalk and other failure modes. In prokaryotes, orthogonal control has often been achieved with the use of orthogonal polymerases (RNAPs), in particular T7 RNAP. Unfortunately, T7 RNAP-mediated expression frequently leads to a drain of cellular resources and associated toxicity, and there is a need for more precise control over such systems. Here we present a universal plasmid system (UPS) for the modular and facile construction of synthetic genetic systems based on orthogonal transcriptional control elements, and simple ‘macros’ that lead to different states of T7 RNA polymerase-based expression. Initially, we have focused on T7 RNAP homeostasis circuits, which produce defined gene expression under a variety of conditions, since both the production and regulation of T7 RNAP are under its own control and insulated against host RNAP regulation. By systematically assembling versions of the homeostasis circuit, we demonstrate that in a variety of Escherichia coli strains – DH10B, MG1655 and the probiotic strain Nissle 1917 – gene expression outputs can be predicted by a simple quantitative model and readily controlled via mutant T7 RNAP promoters. Furthermore, by encoding all circuit elements on a single broad host range vector, the UPS homeostasis system can be ported across a number of industrially and therapeutically relevant species – Pseudomonas putida, Salmonella typhimurium, Serratia marcescens, and Bacillus subtilis – and can be used to constitutively express genes at preset levels based solely on T7 RNAP-mediated control. Further instantiations of additional T7 RNAP-based ‘macros’ should allow the expression of spike, oscillatory and other waveforms of expression.