Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species

Topp, Shana; Reynoso, Colleen M. K.; Seeliger, Jessica C.; Goldlust, Ian S.; Desai, Shawn K.; Murat, Dorothée; Shen, Aimee; Puri, Aaron W.; Komeili, Arash; Bertozzi, Carolyn R.; Scott, June R.; Gallivan, Justin P.

doi:10.1128/aem.00247-11

Cited by 24 publications

(44 citation statements)

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“…We assembled a library of gene expression activators and repressors, functioning at both the RNA and protein levels, with a diversity of expression levels and tested them in both processed and nonprocessed extracts ( Figure 5). The six circuits conditions that we assayed were: small transcription-activating RNAs (STARs) [72]; RNA transcriptional repressors [73]; the inducible protein LuxR transcription factor [74]; CRISPR-Cas9-mediated DNA cleavage [75,76]; toehold switch translation activating RNAs [77]; and a translational theophylline riboswitch [78]. The individual reaction conditions for each circuit are presented in Supplementary Table 1.…”

Section: A Wide Array Of Transcriptional Genetic Circuits Are Functiomentioning

confidence: 99%

Deconstructing cell-free extract preparation forin vitroactivation of transcriptional genetic circuitry

Silverman¹,

Kelley‐Loughnane

Lucks³

et al. 2018

Preprint

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Recent advances in cell-free gene expression (CFE) systems have enabled their use for a host of synthetic biology applications, particularly for rapid prototyping of genetic circuits designed as biosensors. Despite the proliferation of cell-free protein synthesis platforms, the large number of currently existing protocols for making CFE extracts muddles the collective understanding of how the method by which an extract is prepared affects its functionality. Specifically, a key goal toward developing cell-free biosensors based on native genetic regulators is activating the transcriptional machinery present in bacterial extracts for protein synthesis. However, protein yields from genes transcribed in vitro by the native Escherichia coli RNA polymerase are quite low in conventional crude extracts originally optimized for expression by the bacteriophage transcriptional machinery. Here, we show that cell-free expression of genes under bacterial σ 70 promoters is constrained by the rate of transcription in crude extracts and that processing the extract with a ribosomal run-off reaction and subsequent dialysis can alleviate this constraint. Surprisingly, these processing steps only enhance protein synthesis in genes under native regulation, indicating that the translation rate is unaffected. We further investigate the role of other common process variants on extract performance and demonstrate that bacterial transcription is inhibited by including glucose in the growth culture, but is unaffected by flash-freezing the cell pellet prior to lysis. Our final streamlined protocol for preparing extract by sonication generates extract that facilitates expression from a diverse set of sensing modalities including protein and RNA regulators. We anticipate that this work will clarify the methodology for generating CFE extracts that are active for biosensing and will encourage the further proliferation of cell-free gene expression technology for new applications.

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Section: A Wide Array Of Transcriptional Genetic Circuits Are Functiomentioning

confidence: 99%

Deconstructing cell-free extract preparation forin vitroactivation of transcriptional genetic circuitry

Silverman¹,

Kelley‐Loughnane

Lucks³

et al. 2018

Preprint

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“…4b), a strategy predominantly used by prokaryotes to regulate translation initiation (Chang et al, 2012). Based on this mechanism, synthetic riboswitches have been engineered to sense a series of metabolites, including theophylline (Topp et al, 2010), ammeline (Dixon et al, 2010), and thiamine pyrophosphate (Muranaka et al, 2009). In one study, a natural AdoCbl-responsive (also known as coenzyme B 12 ) riboswitch was modified to create synthetic AdoCbl biosensors, which has been suggested to modulate translation by AdoCbl-induced sequestration of the RBS.…”

Section: Biosensors Based On Rnasmentioning

confidence: 99%

Applications and advances of metabolite biosensors for metabolic engineering

Liu

Evans

Zhang

2015

Metabolic Engineering

169

138

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Quantification and regulation of pathway metabolites is crucial for optimization of microbial production bioprocesses. Genetically encoded biosensors provide the means to couple metabolite sensing to several outputs invaluable for metabolic engineering. These include semi-quantification of metabolite concentrations to screen or select strains with desirable metabolite characteristics, and construction of dynamic metabolite-regulated pathways to enhance production. Taking inspiration from naturally occurring systems, biosensor functions are based on highly diverse mechanisms including metabolite responsive transcription factors, two component systems, cellular stress responses, regulatory RNAs, and protein activities. We review recent developments in biosensors in each of these mechanistic classes, with considerations towards how these sensors are engineered, how new sensing mechanisms have led to improved function, and the advantages and disadvantages of each of these sensing mechanisms in relevant applications. We particularly highlight recent examples directly using biosensors to improve microbial production, and the great potential for biosensors to further inform metabolic engineering practices.

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“…Escherichia coli is one of the most widely studied and wellcharacterized model organisms and many tools for synthetic biology, including those based on riboswitches or RNATs, are often created and implemented in E. coli (Neupert et al 2008;Topp et al 2010;Berens and Suess 2015;Hoynes-O'Connor et al 2015;Rodrigues and Rodrigues 2018). This section will discuss a few of the more interesting applications of synthetic riboswitches that have been tested in E. coli, as well as their implications.…”

Section: Escherichia Colimentioning

confidence: 99%

Applications and limitations of regulatoryRNAelements in synthetic biology and biotechnology

et al. 2019

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Summary Synthetic biology requires the design and implementation of novel enzymes, genetic circuits or even entire cells, which can be controlled by the user. RNA‐based regulatory elements have many important functional properties in this regard, such as their modular nature and their ability to respond to specific external stimuli. These properties have led to the widespread exploration of their use as gene regulation devices in synthetic biology. In this review, we focus on two major types of RNA elements: riboswitches and RNA thermometers (RNATs). We describe their general structure and function, before discussing their potential uses in synthetic biology (e.g. in the production of biofuels and biodegradable plastics). We also discuss their limitations, and novel strategies to implement RNA‐based regulatory devices in biotechnological applications. We close with a description of some common model organisms used in synthetic biology, with a focus on the current applications and limitations of RNA‐based regulation.

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Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species

Cited by 24 publications

References 0 publications

Deconstructing cell-free extract preparation forin vitroactivation of transcriptional genetic circuitry

Deconstructing cell-free extract preparation forin vitroactivation of transcriptional genetic circuitry

Applications and advances of metabolite biosensors for metabolic engineering

Applications and limitations of regulatoryRNAelements in synthetic biology and biotechnology

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