Using Spinach Aptamer to Correlate mRNA and Protein Levels in Escherichia coli

Pothoulakis, Georgios; Ellis, Tom

doi:10.1016/bs.mie.2014.10.047

Cited by 5 publications

(6 citation statements)

References 13 publications

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“…Additionally, mathematical modeling indicates that this organization can lead to differences in protein abundance if diffusion is sufficiently slow. To test this, we studied gene expression in vesicles by tracking transcription and translation using a coupled mRNA/protein reporter technique described previously. ,− Briefly, the RNA aptamer Spinach2 was inserted downstream of a gene coding for a red fluorescent protein, mCherry (Figure A). Spinach2 fluoresces in the green range upon hybridization with the fluorophore DFHBI-1T.…”

Section: Resultsmentioning

confidence: 99%

Crowding-Induced Spatial Organization of Gene Expression in Cell-Sized Vesicles

Chauhan¹,

Norred²,

Simpson³

et al. 2022

ACS Synth. Biol.

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Cell-free protein synthesis is an important tool for studying gene expression and harnessing it for applications. In cells, gene expression is regulated in part by the spatial organization of transcription and translation. Unfortunately, current cell-free approaches are unable to control the organization of molecular components needed for gene expression, which limits the ability to probe and utilize its effects. Here, we show, using complementary computational and experimental approaches, that macromolecular crowding can be used to control the spatial organization and translational efficiency of gene expression in cell-sized vesicles. Computer simulations and imaging experiments reveal that, as crowding is increased, DNA plasmids become localized at the inner surface of vesicles. Ribosomes, in contrast, remain uniformly distributed, demonstrating that crowding can be used to differentially organize components of gene expression. We further carried out cell-free protein synthesis reactions in cell-sized vesicles and quantified mRNA and protein abundance. At sufficiently high levels of crowding, we observed localization of mRNA near vesicle surfaces, a decrease in translational efficiency and protein abundance, and anomalous scaling of protein abundance as a function of vesicle size. These results are consistent with high levels of crowding causing altered spatial organization and slower diffusion. Our work demonstrates a straightforward way to control the organization of gene expression in cell-sized vesicles and provides insight into the spatial regulation of gene expression in cells.

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Section: Resultsmentioning

confidence: 99%

Crowding-Induced Spatial Organization of Gene Expression in Cell-Sized Vesicles

Chauhan¹,

Norred²,

Simpson³

et al. 2022

ACS Synth. Biol.

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“…To understand how the combination of crowding and confinement affects gene expression, we performed cell-free protein synthesis (CFPS) reactions in vesicles crowded with Ficoll-70. Transcription and translation were tracked simultaneously using a coupled mRNA/protein reporter technique described in previous work 9,[22][23][24] . Briefly, Spinach2 25 , an RNA aptamer which fluoresces in the green range upon hybridization with the fluorophore DFHBI-1T, was inserted downstream of a gene coding for a red fluorescent protein, mCherry 26 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Synergistic interactions between confinement and macromolecular crowding spatially order transcription and translation in cell-free expression

Norred

Chauhan

et al. 2018

Preprint

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Synergistic interactions between macromolecular crowding and confinementspatially organize transcription and translation in cells. Yet, reproducing such spatial ordering in cell-free expression platforms has proven to be elusive. Here we report crowding-and confinement-driven spatial self-organization of cell-free expression that mimics expression behavior within and around the nucleoid of prokaryotes. These experiments use Ficoll-70 to approximate cellular macromolecular crowding conditions within cell-size lipid vesicles. Intriguingly, there was an abrupt change in transcriptional dynamics when crowding reached physiologically relevant levels. Imaging experiments revealed that this change in transcriptional dynamics was coincident with localization of plasmid DNA and mRNA at the vesicle wall. Computer simulations demonstrated that crowding leads to an entropically induced attraction between plasmid DNA and the wall, causing localization of DNA near the wall at sufficiently high crowding levels. The experiments demonstrate cell-like spatial organization of translation, where translational activity is controlled by chromosomally-templated positioning of mRNA. This cell-free system provides a flexible experimental platform to probe the underlying mechanisms of self-organization of membrane-less structures in cells and the spatial control of gene expression.

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“…Since then, spinach was used for in vitro visualization of enzymatic RNA synthesis (Höfer et al, 2013 ), conformational transitions of a cyclic-di-GMP riboswitch (Luo et al, 2014b ), and siRNA processing by dicer (Rogers et al, 2015 ). In combination with the expression of fluorescent proteins, RNA-spinach constructs allowed the visualization and quantification of transcription and translation in bacteria (Pothoulakis and Ellis, 2015 ) and artificial cells (van Nies et al, 2015 ).…”

Section: Small Molecule Aptasensorsmentioning

confidence: 99%

Selection and Biosensor Application of Aptamers for Small Molecules

2016

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Small molecules play a major role in the human body and as drugs, toxins, and chemicals. Tools to detect and quantify them are therefore in high demand. This review will give an overview about aptamers interacting with small molecules and their selection. We discuss the current state of the field, including advantages as well as problems associated with their use and possible solutions to tackle these. We then discuss different kinds of small molecule aptamer-based sensors described in literature and their applications, ranging from detecting drinking water contaminations to RNA imaging.

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Using Spinach Aptamer to Correlate mRNA and Protein Levels in Escherichia coli

Cited by 5 publications

References 13 publications

Crowding-Induced Spatial Organization of Gene Expression in Cell-Sized Vesicles

Crowding-Induced Spatial Organization of Gene Expression in Cell-Sized Vesicles

Synergistic interactions between confinement and macromolecular crowding spatially order transcription and translation in cell-free expression

Selection and Biosensor Application of Aptamers for Small Molecules

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