Development of a 2,4-Dinitrotoluene-Responsive Synthetic Riboswitch in E. coli cells

Davidson, Molly E.; Harbaugh, Svetlana; Chushak, Yaroslav; Stone, Morley O.; Kelley‐Loughnane, Nancy

doi:10.21236/ada570108

Cited by 11 publications

(16 citation statements)

References 10 publications

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“…5) indicate that at least semi-quantitative detection at sub mmol l -1 concentrations are possible. The fold change and limits of detection are similar to reports for a 2,4-Dinitrotoluene (DNT) responsive riboswitch which showed an activation ratio up to 9.8 (Davidson et al, 2013). This is similar to the 11-fold change in fluorescence observed with Clone 4 when exposed to 44 mmol l -1 RDX (Fig.…”

Section: Discussionsupporting

confidence: 85%

“…The RDX riboswitch was sensitive to as low as 0.4 mmol l -1 and the response was saturated by 4.4 mmol l -1 . In contrast, the DNT riboswitch had a detectable increase in fluorescence at around 50 mmol l -1 (Davidson et al, 2013).…”

Section: Discussionmentioning

confidence: 80%

“…Development of riboswitches with the desired functionality generally consists of screen-ing large clone libraries containing random linker sequences. The length and sequence of the linker determines the structure of the riboswitch which, in turn, determines its ability to regulate the target gene (Davidson et al, 2013;Lynch et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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Detection of hexahydro-1,3-5-trinitro-1,3,5-triazine (RDX) with a microbial sensor

Eberly

Mayo

Carr

et al. 2019

J. Gen. Appl. Microbiol.

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Section: Discussionsupporting

confidence: 85%

Section: Discussionmentioning

confidence: 80%

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Detection of hexahydro-1,3-5-trinitro-1,3,5-triazine (RDX) with a microbial sensor

Eberly

Mayo

Carr

et al. 2019

J. Gen. Appl. Microbiol.

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“…So far, in vivo active riboswitches rely on a small set of ligands – theophylline, tetracycline, neomycin [53], 2,4‐dinitrotoluene [107], ammeline, 5‐aza‐cytosine [108] or pyrimido[4,5‐ d ]pyrimidine‐2,4‐diamine (PPDA) [43], although several dozen small molecule‐binding aptamers have been selected by a process called SELEX (systematic evolution of ligands by exponential enrichment), an in vitro technology invented about a decade before the discovery of natural riboswitches [109, 110]. During SELEX, a huge combinatorial library of RNA (and sometimes DNA) molecules is subjected to affinity of a small RNA chromatography to the ligand of choice.…”

Section: What Makes a Riboswitch Out Of An Aptamer?mentioning

confidence: 99%

RNA aptamers as genetic control devices: The potential of riboswitches as synthetic elements for regulating gene expression

Berens

Groher

Suess

2015

Biotechnology Journal

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RNA utilizes many different mechanisms to control gene expression. Among the regulatory elements that respond to external stimuli, riboswitches are a prominent and elegant example. They consist solely of RNA and couple binding of a small molecule ligand to the so-called "aptamer domain" with a conformational change in the downstream "expression platform" which then determines system output. The modular organization of riboswitches and the relative ease with which ligand-binding RNA aptamers can be selected in vitro against almost any molecule have led to the rapid and widespread adoption of engineered riboswitches as artificial genetic control devices in biotechnology and synthetic biology over the past decade. This review highlights proof-of-principle applications to demonstrate the versatility and robustness of engineered riboswitches in regulating gene expression in pro- and eukaryotes. It then focuses on strategies and parameters to identify aptamers that can be integrated into synthetic riboswitches that are functional in vivo, before finishing with a reflection on how to improve the regulatory properties of engineered riboswitches, so that we can not only further expand riboswitch applicability, but also finally fully exploit their potential as control elements in regulating gene expression.

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“…These selected colonies were then grown up as clonal populations in 96-well plates and tested in media in the presence and absence of the ligand, to identify clones that showed increased β-galactosidase activity in the presence of ligand. Similar plate-based screens of clonal populations harboring RNA switch libraries have been shown with other reporter proteins encoding fluorescent proteins (Klauser et al 2012;Davidson et al 2013). However, the selection of an enzymatic reporter protein like β-galactosidase limits the throughput of the activity screen to clonal, plate-based assays, which show a lower throughput (several thousand constructs at most) than reporter proteins that are compatible with assays that enable the measurement and subsequent isolation of single cells in mixed populations, such as fluorescence-activated cell sorting (FACS).…”

Section: High-throughput Screening Of Rna Switch Libraries Allows Formentioning

confidence: 59%

RNA Switches for Synthetic Biology

Schmidt

Smolke

2019

Cold Spring Harb Perspect Biol

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In addition to coding for protein sequences, RNA molecules encode a diverse set of generegulatory elements. RNA switches are one class of gene-regulatory elements that control protein expression in a manner that is dependent on the concentration of specific ligand molecules. These allosteric gene-regulatory elements have been shown as useful tools in engineering diverse cell types to display novel function. In particular, RNA switches have been used as genetically encoded biosensors and conditional controllers to direct cellular decisions based on the system's changing environment. A significant focus in the field has been the generation of novel RNA switches that are tailored for different biological systems. We review approaches that have been used to generate RNA switches, which leverage the unique physical properties of RNA and the myriad ways in which RNA can modulate gene expression.Outline 1 Naturally occurring riboswitches can serve as a starting point for designing engineered RNA switches 2 RNA switches can be rationally designed using rules-based approaches 3 Computational tools enable a more efficient RNA switch design process 4 High-throughput screening of RNA switch libraries allows for generation of highly functional switch sequences 5 Conclusions ReferencesEditors:

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Development of a 2,4-Dinitrotoluene-Responsive Synthetic Riboswitch in E. coli cells

Cited by 11 publications

References 10 publications

Detection of hexahydro-1,3-5-trinitro-1,3,5-triazine (RDX) with a microbial sensor

Detection of hexahydro-1,3-5-trinitro-1,3,5-triazine (RDX) with a microbial sensor

RNA aptamers as genetic control devices: The potential of riboswitches as synthetic elements for regulating gene expression

RNA Switches for Synthetic Biology

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