Multi-input regulation and logic with T7 promoters in cells and cell free systems

Journal Article · Wed Jan 01 00:00:00 EST 2014 · ACS Synthetic Biology

DOI:https://doi.org/10.1021/sb4000756· OSTI ID:1134635

Iyer, Sukanya ^[1]; Karig, David K ^[1]; Norred, Sarah E ^[1]; Simpson, Michael L ^[1]; Doktycz, Mitchel John ^[1]

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1. ORNL

Engineered gene circuits offer an opportunity to harness biological systems for biotechnological and biomedical applications. However, reliance on host E. coli promoters for the construction of circuit elements, such as logic gates, makes 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 a commercially available E. coli cell-free protein expression system. Together, our results contribute to the first demonstration of multi-input regulation of T7 promoters and expand the utility of T7 promoters in cell based as well as cell-free gene circuits.

• Research Organization: Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Nanophase Materials Sciences (CNMS)
• Sponsoring Organization: Work for Others (WFO)
• DOE Contract Number: DE-AC05-00OR22725
• OSTI ID: 1134635
• Journal Information: ACS Synthetic Biology, Vol. 3, Issue 6; ISSN 2161--5063
• Country of Publication: United States
• Language: English