Thrombin-Mediated Transcriptional Regulation Using DNA Aptamers in DNA-Based Cell-Free Protein Synthesis

Realizing the potential of cell-free systems will require development of ligand-sensitive gene promoters that control gene expression in response to a ligand of interest. Here, we describe an approach to designing ligand-sensitive transcriptional control in cell-free systems that is based on the combination of a DNA aptamer that binds thrombin and the T7 bacteriophage promoter. Placement of the aptamer near the T7 promoter, and using a primarily single-stranded template, results in up to a 6-fold change in gene expression in a ligand concentration-dependent manner. We further demonstrate that the sensitivity to thrombin concentration and the fold change in expression can be tuned by altering the position of the aptamer. The results described here pave the way for the use of DNA aptamers to achieve modular regulation of transcription in response to a wide variety of ligands in cell-free systems

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

<div><p>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 <i>E. coli</i> cell-free protein expression system. Our results expand the utility of T7 promoters in cell based as well as cell-free synthetic biology applications.</p> </div

Effect of auxiliary operators on LacI mediated repression of T7lacO promoters (in vivo).

<p>A) illustrates promoter sequences containing T7lacO promoters with auxiliary operator sequences of different strengths. B) Protein expression responses to 30 μM IPTG from the constructs depicted in A). GFP concentration units are expressed as µM/OD₆₀₀. C) Dose responses to IPTG from the different constructs. Fluorescence response values are normalized to cell counts as determined by optical density values. Error bars depict standard deviation of triplicate measurements. Lines depict nonlinear regression fits to the Hill equation.</p

Design strategy for achieving combinatorial regulation of expression from T7 promoters.

<p>A) An auxiliary lacO is placed upstream to a conventional T7lacO promoter to create stronger LacI repressible T7 promoters. DNA looping is induced by the binding of a single LacI tetramer to both of the lacO binding sites. B) TetR binding regions (tetO) placed within this DNA looping framework, at regions indicated by grey box, can enable multi-input regulation by interfering with LacI mediated looping.</p

Effect of tetO on LacI mediated repression of T7lacO when tetO is in between the two lac operators in cell free systems.

<p>A) Fluorescence response from pDRT7 77 to LacI and TetR proteins. B) Shows fluorescence response from pDRT7 14 and pDRT7 77 plasmids to presence of 300 μM IPTG and/or 200ng/ml aTc. Error bars in the figure depict standard deviations of triplicate measurements.</p