Multi-Input Regulation and Logic with T7 Promoters in Cells and Cell-Free Systems

Sukanya Iyer,David K Karig,Mitchel J Doktycz,S Elizabeth Norred,Michael L Simpson,Mark Isalan

doi:10.1371/journal.pone.0078442

Sukanya Iyer, David K Karig + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0078442

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Abstract

Engineered gene circuits offer an opportunity to harness biological systems for biotechnological and biomedical applications. However, reliance on native host promoters for the construction of circuit elements, such as logic gates, can make the implementation of predictable, independently functioning circuits difficult. In contrast, T7 promoters offer a simple orthogonal expression system for use in a variety of cellular backgrounds and even in cell-free systems. Here we develop a T7 promoter system that can be regulated by two different transcriptional repressors for the construction of a logic gate that functions in cells and in cell-free systems. We first present LacI repressible T7lacO promoters that are regulated from a distal lac operator site for repression. We next explore the positioning of a tet operator site within the T7lacO framework to create T7 promoters that respond to tet and lac repressors and realize an IMPLIES gate. Finally, we demonstrate that these dual input sensitive promoters function in an E. coli cell-free protein expression system. Our results expand the utility of T7 promoters in cell based as well as cell-free synthetic biology applications.

Highlights

Engineering synthetic gene circuits entails the redesign of existing gene networks or the creation of novel genetic functions to perform a predetermined task
We examine the effect of placement of tet operator (tetO), the binding site for the TetR protein, into the Lac repressor proteins (LacI) looping framework so as to generate T7 promoters that respond to both TetR and LacI (Figure 1)
We demonstrate the functionality of these TetR and LacI repressible T7 promoters in both live E. coli cells and in cell-free expression systems

Summary

Introduction

Engineering synthetic gene circuits entails the redesign of existing gene networks or the creation of novel genetic functions to perform a predetermined task. Construction of these circuits has been valuable in attaining a bottom up understanding of biological systems[1] and offers potential for harnessing biological function for biotechnology[2,3] and biomedicine[4,5]. Well-characterized genetic components have been integrated into circuits that function as logic gates [6,7], memory elements, clocks[8] and counters. Despite the fact that a rapidly growing number of gene circuits are being published, the complexity of the systems is not keeping pace[11]. Orthogonal expression systems that insulate the synthetic gene circuits from other biological networks are required[12,13,14]

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