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Fluorescent protein-based FRET sensor for intracellular monitoring of redox status in bacteria at single cell level

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Fluorescent protein-based FRET sensor for intracellular monitoring of redox status in bacteria at single cell level. / Abraham, Bobin George; Santala, Ville; Tkachenko, Nikolai V.; Karp, Matti.

In: Analytical and Bioanalytical Chemistry, Vol. 406, No. 28, 2014, p. 7195-7204.

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Abraham, Bobin George ; Santala, Ville ; Tkachenko, Nikolai V. ; Karp, Matti. / Fluorescent protein-based FRET sensor for intracellular monitoring of redox status in bacteria at single cell level. In: Analytical and Bioanalytical Chemistry. 2014 ; Vol. 406, No. 28. pp. 7195-7204.

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@article{7845ca1359e9464c8d594dbb167e500c,
title = "Fluorescent protein-based FRET sensor for intracellular monitoring of redox status in bacteria at single cell level",
abstract = "Monitoring of intracellular redox status in a bacterial cell provides vital information about the physiological status of the cell, which can be exploited in several applications such as metabolic engineering and computational modeling. Fluorescent protein-based genetically encoded sensors can be used to monitor intracellular oxidation/reduction status. This study reports the development of a redox sensor for intracellular measurements using fluorescent protein pairs and the phenomenon of F{\"o}rster resonance energy transfer (FRET). For the development of the sensor, fluorescent proteins Citrine and Cerulean were genetically modified to carry reactive cysteine residues on the protein surface close to the chromophore and a constructed FRET pair was fused using a biotinylation domain as a linker. In oxidized state, the FRET pairs are in close proximity by labile disulfide bond formation resulting in higher FRET efficiency. In reducing environment, the FRET is diminished due to the increased distance between FRET pairs providing large dynamic measurement range to the sensor. Intracellular studies in Escherichia coli mutants revealed the capability of the sensor in detecting real-time redox variations at single cell level. The results were validated by intensity based and time resolved measurements. The functional immobilization of the fluorescent protein-based FRET sensor at solid surfaces for in vitro applications was also demonstrated. [Figure not available: see fulltext.]",
author = "Abraham, {Bobin George} and Ville Santala and Tkachenko, {Nikolai V.} and Matti Karp",
note = "Published online: 16 September 2014<br/>Contribution: organisation=keb,FACT1=1<br/>Portfolio EDEND: 2014-09-22<br/>Publisher name: Springer; Fachgruppe Analytische Chemie",
year = "2014",
doi = "10.1007/s00216-014-8165-1",
language = "English",
volume = "406",
pages = "7195--7204",
journal = "Analytical and Bioanalytical Chemistry",
issn = "1618-2642",
publisher = "Springer Verlag",
number = "28",

}

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TY - JOUR

T1 - Fluorescent protein-based FRET sensor for intracellular monitoring of redox status in bacteria at single cell level

AU - Abraham, Bobin George

AU - Santala, Ville

AU - Tkachenko, Nikolai V.

AU - Karp, Matti

N1 - Published online: 16 September 2014<br/>Contribution: organisation=keb,FACT1=1<br/>Portfolio EDEND: 2014-09-22<br/>Publisher name: Springer; Fachgruppe Analytische Chemie

PY - 2014

Y1 - 2014

N2 - Monitoring of intracellular redox status in a bacterial cell provides vital information about the physiological status of the cell, which can be exploited in several applications such as metabolic engineering and computational modeling. Fluorescent protein-based genetically encoded sensors can be used to monitor intracellular oxidation/reduction status. This study reports the development of a redox sensor for intracellular measurements using fluorescent protein pairs and the phenomenon of Förster resonance energy transfer (FRET). For the development of the sensor, fluorescent proteins Citrine and Cerulean were genetically modified to carry reactive cysteine residues on the protein surface close to the chromophore and a constructed FRET pair was fused using a biotinylation domain as a linker. In oxidized state, the FRET pairs are in close proximity by labile disulfide bond formation resulting in higher FRET efficiency. In reducing environment, the FRET is diminished due to the increased distance between FRET pairs providing large dynamic measurement range to the sensor. Intracellular studies in Escherichia coli mutants revealed the capability of the sensor in detecting real-time redox variations at single cell level. The results were validated by intensity based and time resolved measurements. The functional immobilization of the fluorescent protein-based FRET sensor at solid surfaces for in vitro applications was also demonstrated. [Figure not available: see fulltext.]

AB - Monitoring of intracellular redox status in a bacterial cell provides vital information about the physiological status of the cell, which can be exploited in several applications such as metabolic engineering and computational modeling. Fluorescent protein-based genetically encoded sensors can be used to monitor intracellular oxidation/reduction status. This study reports the development of a redox sensor for intracellular measurements using fluorescent protein pairs and the phenomenon of Förster resonance energy transfer (FRET). For the development of the sensor, fluorescent proteins Citrine and Cerulean were genetically modified to carry reactive cysteine residues on the protein surface close to the chromophore and a constructed FRET pair was fused using a biotinylation domain as a linker. In oxidized state, the FRET pairs are in close proximity by labile disulfide bond formation resulting in higher FRET efficiency. In reducing environment, the FRET is diminished due to the increased distance between FRET pairs providing large dynamic measurement range to the sensor. Intracellular studies in Escherichia coli mutants revealed the capability of the sensor in detecting real-time redox variations at single cell level. The results were validated by intensity based and time resolved measurements. The functional immobilization of the fluorescent protein-based FRET sensor at solid surfaces for in vitro applications was also demonstrated. [Figure not available: see fulltext.]

UR - http://www.scopus.com/inward/record.url?scp=84919725200&partnerID=8YFLogxK

U2 - 10.1007/s00216-014-8165-1

DO - 10.1007/s00216-014-8165-1

M3 - Article

VL - 406

SP - 7195

EP - 7204

JO - Analytical and Bioanalytical Chemistry

JF - Analytical and Bioanalytical Chemistry

SN - 1618-2642

IS - 28

ER -