Supplementary Information from Preservation of protein expression systems at elevated temperatures for portable therapeutic production

Many biotechnology capabilities are limited by stringent storage needs of reagents, largely prohibiting use outside of specialized laboratories. Focusing on a large class of protein-based biotechnology applications, we address this issue by developing a method for preserving cell-free protein expression systems for months above room temperature. Our approach realizes unprecedented long-term stability at elevated temperatures by leveraging the sugar alcohol trehalose, a simple, low-cost, open-air drying step, and strategic separation of reaction components during drying. The resulting preservation capacity enables efficient production of a wide range of on-demand proteins under adverse conditions, for instance during emergency outbreaks or in remote locations. To demonstrate application potential, we use cell-free reagents subjected to months of exposure at 37°C and atmospheric conditions to produce sufficient concentrations of a pyocin protein to kill <i>Pseudomonas aeruginosa</i>, a troublesome pathogen for traumatic and burn wound injuries. Our work makes possible new biotechnology applications that demand ruggedness and scalability

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