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 tetO on LacI mediated repression of T7lacO when tetO is in between the two lac operators (in vivo).

<p>Shown in (A) are the plasmid constructs pDRT7 14 and pDRT7 77. B) Displays the responses of these plasmids to presence /absence of 30 μM IPTG and 200 ng/ml aTc. C) Gene expression response, as determined by the normalized fluorescence response, of the pDRT7 77 plasmid to a range of IPTG and aTc concentrations. aTc concentration (ng/mL) is displayed on the X axis and the Y-axis denotes IPTG concentrations (μM). GFP fluorescence measurements in B and C are expressed as µM/OD₆₀₀. D) is a schematic of the IMPLIES logic gate realized using the pDRT7 77 plasmid. Error bars depict standard deviation of triplicate measurements.</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

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

Resource Sharing Controls Gene Expression Bursting

Episodic gene expression, with periods of high expression separated by periods of no expression, is a pervasive biological phenomenon. This bursty pattern of expression draws from a finite reservoir of expression machinery in a highly time variant way, i.e., requiring no resources most of the time but drawing heavily on them during short intense bursts, that intimately links expression bursting and resource sharing. Yet, most recent investigations have focused on specific molecular mechanisms intrinsic to the bursty behavior of individual genes, while little is known about the interplay between resource sharing and global expression bursting behavior. Here, we confine Escherichia coli cell extract in both cell-sized microfluidic chambers and lipid-based vesicles to explore how resource sharing influences expression bursting. Interestingly, expression burst size, but not burst frequency, is highly sensitive to the size of the shared transcription and translation resource pools. The intriguing implication of these results is that expression bursts are more readily amplified than initiated, suggesting that burst formation occurs through positive feedback or cooperativity. When extrapolated to prokaryotic cells, these results suggest that large translational bursts may be correlated with large transcriptional bursts. This correlation is supported by recently reported transcription and translation bursting studies in E. coli. The results reported here demonstrate a strong intimate link between global expression burst patterns and resource sharing, and they suggest that bursting plays an important role in optimizing the use of limited, shared expression resources