A cell-free biosensor for the detection of transcriptional inducers using firefly luciferase as a reporter

Pellinen, Teijo; Huovinen, Tuomas; Karp, Matti

doi:10.1016/j.ab.2004.03.064

Cited by 72 publications

(51 citation statements)

References 20 publications

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“…Importantly, P mer promoter is noticeably leaky in the absence of MerR protein. Thus, MerR protein needs to be present within the TX-TL reaction before the sensor plasmid is added to avoid high background expression of the reporter (LacZ in this case) (36). We therefore pre-expressed MerR protein from its native promoter directly within the TX-TL reactions based on freeze-thaw autolysate (45 minutes, 30°C).…”

Section: Resultsmentioning

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: Resultsmentioning

confidence: 99%

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

et al. 2017

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“…Even simple feedback constructs have not been previously demonstrated in cell-free protein expression systems, despite their functional importance and widespread utilization in live cell gene networks. Also, whereas addition of T7 lysozyme has been used to tune T7 transcription in the past (56), our system is autoregulatory and will be useful in applications such as cell-free biosensors (57). …”

Section: Discussionmentioning

confidence: 99%

Expression optimization and synthetic gene networks in cell-free systems

Karig

Iyer

Simpson

et al. 2011

Nucleic Acids Research

117

124

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Synthetic biology offers great promise to a variety of applications through the forward engineering of biological function. Most efforts in this field have focused on employing living cells, yet cell-free approaches offer simpler and more flexible contexts. Here, we evaluate cell-free regulatory systems based on T7 promoter-driven expression by characterizing variants of TetR and LacI repressible T7 promoters in a cell-free context and examining sequence elements that determine expression efficiency. Using the resulting constructs, we then explore different approaches for composing regulatory systems, leading to the implementation of inducible negative feedback in Escherichia coli extracts and in the minimal PURE system, which consists of purified proteins necessary for transcription and translation. Despite the fact that negative feedback motifs are common and essential to many natural and engineered systems, this simple building block has not previously been implemented in a cell-free context. As a final step, we then demonstrate that the feedback systems developed using our cell-free approach can be implemented in live E. coli as well, illustrating the potential for using cell-free expression to fast track the development of live cell systems in synthetic biology. Our quantitative cell-free component characterizations and demonstration of negative feedback embody important steps on the path to harnessing biological function in a bottom-up fashion.

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“…Thus, biological materials with high sensitivity and selectivity towards toxic metals could be functional as sensing elements when their response signals would be converted to electrical signals by transducers. Functions of toxic metal-responsive transcriptional switch are also fundamental for the whole cell-based (Hynninen and Virta, 2010) and biopolymer-based (Nakanishi et al, 2013;Pellinen et al, 2004) toxic metal sensors.…”

Section: Introductionmentioning

confidence: 99%

Mercury (II) sensor based on monitoring dissociation rate of the trans-acting factor MerR from cis-element by surface plasmon resonance

Taniguchi

Siddiki

Ueda

et al. 2015

Biosensors and Bioelectronics

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A cell-free biosensor for the detection of transcriptional inducers using firefly luciferase as a reporter

Cited by 72 publications

References 20 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

Expression optimization and synthetic gene networks in cell-free systems

Mercury (II) sensor based on monitoring dissociation rate of the trans-acting factor MerR from cis-element by surface plasmon resonance

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