Portable, On-Demand Biomolecular Manufacturing

Pardee, Keith; Slomovic, Shimyn; Nguyen, Peter Q.; Lee, Jeong Wook; Donghia, Nina M.; Burrill, Devin R.; Ferrante, Tom; McSorley, Fern R.; Furuta, Yoshikazu; Vernet, Andyna; Lewandowski, Michael; Boddy, Christopher N.; Joshi, Neel; Collins, James J.

doi:10.1016/j.cell.2016.09.013

Cited by 314 publications

(299 citation statements)

References 65 publications

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“…Cell-free expression systems, freeze-dried on paper or other supports, have been recently used to create robust biosensors and in situ bioproduction reagents capable of room temperature storage for extended periods of time (5–7, 20). Upon rehydration, such freeze-dried CFE systems support transcription-translation reactions enabling biosensing or bioproduction programmed by the supplied synthetic gene circuits.…”

Section: Introductionmentioning

confidence: 99%

Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression

et al. 2017

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Cell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biology and biotechnology applications, including production of robust field-ready biosensors. Here, we combine programmed cellular autolysis with a freeze-thaw or freeze-dry cycle to create a practical, reproducible, and a labor- and cost-effective approach for rapid production of bacterial lysates for cell-free gene expression. Using this method, robust and highly active bacterial cell lysates can be produced without specialized equipment at a wide range of scales, making cell-free gene expression easily and broadly accessible. Moreover, live autolysis strain can be freeze-dried directly and subsequently lysed upon rehydration to produce active lysate. We demonstrate the utility of autolysates for synthetic biology by regulating protein production and degradation, implementing quorum sensing, and showing quantitative protection of linear DNA templates by GamS protein. To allow versatile and sensitive β-galactosidase (LacZ) based readout we produce autolysates with no detectable background LacZ activity and use them to produce sensitive mercury(II) biosensors with LacZ-mediated colorimetric and fluorescent outputs. The autolysis approach can facilitate wider adoption of cell-free technology for cell-free gene expression as well as other synthetic biology and biotechnology applications, such as metabolic engineering, natural product biosynthesis, or proteomics.

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Section: Introductionmentioning

confidence: 99%

Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression

et al. 2017

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“…months at 37°C) [13,14]. More recently, Pardee et al took a major step towards fieldable protein expression systems when they lyophilized cell-free reagents and used them to both implement biosensor gene networks capable of detecting Ebola [15] and Zika viruses [16] and also produce a wide array of therapeutics [17]. Although the Pardee et al methods demonstrate stabilization at room temperature , they still do not fully address the ruggedness needs for many applications.…”

Section: Introductionmentioning

confidence: 99%

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

Karig

Bessling

Thielen

et al. 2017

J. R. Soc. Interface.

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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 Pseudomonas aeruginosa, a troublesome pathogen for traumatic and burn wound injuries. Our work makes possible new biotechnology applications that demand ruggedness and scalability.

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“…The cell-free TXTL technology has become versatile enough to be employed for biomanufacturing and medicine (Ng et al 2012; Pardee et al 2016), metabolic engineering (Dudley et al 2014), and quantitative disciplines such as biophysics (Tayar A. 2015).…”

mentioning

confidence: 99%

Short DNA containing χ sites enhances DNA stability and gene expression in E. coli cell‐free transcription–translation systems

Marshall

Maxwell

Collins

et al. 2017

Biotech & Bioengineering

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E. coli cell-free transcription-translation (TXTL) systems offer versatile platforms for advanced biomanufacturing and for prototyping synthetic biological parts and devices. Production and testing could be accelerated with the use of linear DNA, which can be rapidly and cheaply synthesized. However, linear DNA is efficiently degraded in TXTL preparations from Escherichia coli. Here, we show that double-stranded DNA encoding χ sites–eight base-pair sequences preferentially bound by the RecBCD recombination machinery–stabilizes linear DNA and greatly enhances the TXTL-based expression and activity of a fluorescent reporter gene, simple regulatory cascades, and T7 bacteriophage particles. The χ-site DNA and the DNA-binding λ protein Gam yielded similar enhancements, and DNA with as few as four χ sites was sufficient to ensure robust gene expression in TXTL. Given the affordability and scalability of producing the short χ-site DNA, this generalized strategy is expected to advance the broad use of TXTL systems across its many applications.

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Portable, On-Demand Biomolecular Manufacturing

Cited by 314 publications

References 65 publications

Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression

Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression

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

Short DNA containing χ sites enhances DNA stability and gene expression in E. coli cell‐free transcription–translation systems

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