Modular control of multiple pathways using engineered orthogonal T7 polymerases

Abstract

Synthetic genetic sensors and circuits enable programmable control over the timing and conditions of gene expression. They are being increasingly incorporated into the control of complex, multigene pathways and cellular functions. Here, we propose a design strategy to genetically separate the sensing/circuitry functions from the pathway to be controlled. This separation is achieved by having the output of the circuit drive the expression of a polymerase, which then activates the pathway from polymerase-specific promoters. The sensors, circuits and polymerase are encoded together on a ‘controller’ plasmid. Variants of T7 RNA polymerase that reduce toxicity were constructed and used as scaffolds for the construction of four orthogonal polymerases identified via part mining that bind to unique promoter sequences. This set is highly orthogonal and induces cognate promoters by 8- to 75-fold more than off-target promoters. These orthogonal polymerases enable four independent channels linking the outputs of circuits to the control of different cellular functions. As a demonstration, we constructed a controller plasmid that integrates two inducible systems, implements an AND logic operation and toggles between metabolic pathways that change Escherichia coli green (deoxychromoviridans) and red (lycopene). The advantages of this organization are that (i) the regulation of the pathway can be changed simply by introducing a different controller plasmid, (ii) transcription is orthogonal to host machinery and (iii) the pathway genes are not transcribed in the absence of a controller and are thus more easily carried without invoking evolutionary pressure.

Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants

“…The bulk of these studies were done in vitro, and quantification of T7 RNAP activity was done by northern blotting, which, although providing a direct measure of transcriptional activity of a T7 expression system, does not give an accurate depiction of in vivo activity. A more recent study found that mutating the specificity loop according to multisequence alignment with distant T7 RNAP homologs significantly alters the specificity of full-length T7 in vivo (29). Here, we propose that mutations of a single amino acid are sufficient for modifying the specificity of T7 RNAP to create orthogonal expression systems using split T7 RNAP.…”

Modularity, context-dependence, and insulation in engineered biological circuits

“…As far as transcriptional resources are concerned, RNA polymerase from phage T7 allows, to some extent, to decouple transcription from the host resources [59]. More recently, several other RNA polymerases have been identified from other phages or from directed evolution experiments [60,61,62]. Due to the high transcription e ciency of these polymerases, toxicity is often an issue and hence their concentration should be limited.…”

Mammalian synthetic biology: emerging medical applications

“…Phage bio-parts are orthogonal in both prokaryotic and eukaryotic cells and their robustness has been demonstrated in the past decades through their ubiquitous presence in common laboratory protocols. Specifically, the T7 promoter has been extensively used in synthetic biology due to its ability to drive high-level gene expression in an orthogonal, low-toxicity fashion [160][161][162]. Additionally, phage-occurring recombinases such as Cre, PhiC31 and Bxb1 have been widely used in the construction of several gene networks, including Boolean logic gates and counters [163,164].…”