Peer Reviewed: Applications of Reporter Genes

Lewis, John L.; Feltus, Agatha; Ensor, Mark; Ramanathan, Sridhar; Daunert, Sylvia

doi:10.1021/ac9819638

Cited by 69 publications

(47 citation statements)

References 28 publications

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“…1,2) b-Gal is considered as one of the most important glycoside hydrolases due to its various applications in dairy and food industries, and in genetic engineering. [3][4][5][6][7][8][9] In plants, numerous studies of b-Gal have been done mainly related to fruit ripening, [10][11][12][13][14][15] and plant growth and development. [16][17][18][19][20][21][22][23][24] Most of the research on b-Gal in plants has been done on dicotyledons due to the larger amount of pectic polysaccharide than in monocotyledons, so that research of b-Gal from monocotyledons is very small.…”

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confidence: 99%

Purification and Characterization of Extracellular β-Galactosidase Secreted by Supension Cultured Rice (Oryza sativaL.) Cells

Kaneko

Kobayashi

2003

Bioscience, Biotechnology, and Biochemistry

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A b-galactosidase was puriˆed 1300-fold by lactosylSepharose 4B and Sephacryl S-200 column chromatographies from the cultured medium of a rice-cell suspension. The puriˆed enzyme appeared as 47 kD and 40 kD polypeptides on SDS-PAGE and had a speciˆc activity of 65.1 units W mg. Optimum activity was observed at pH 3.5 and 609 C. The enzyme released galactose from galactoxyloglucan and pectic galactans.

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confidence: 99%

Purification and Characterization of Extracellular β-Galactosidase Secreted by Supension Cultured Rice (Oryza sativaL.) Cells

Kaneko

Kobayashi

2003

Bioscience, Biotechnology, and Biochemistry

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“…Reporter genes are often coupled to a regulatory element that recognizes an analyte and confers selectivity to the system, while the reporter protein produces a detectable signal controlling the sensitivity of the system (Lewis et al, 1998;Naylor, 1999;Wood, 1995). To date, several types of proteins have been employed as reporter proteins in cell-based biosensing systems (Daunert et al, 2000;Kohler et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Novel reporter gene in a fluorescent‐based whole cell sensing system

Feliciano

Liu

Daunert

2006

Biotech & Bioengineering

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A common problem encountered when using fluorescence detection in real samples analysis is that the matrix may contain compounds that autofluorescence or that can be excited at the wavelengths of commonly employed fluorescent reporter molecules. This causes an increase in background fluorescence, which in turn tends to compromise the detection limits of the system. To address this issue, we investigated the use of a reporter enzyme that produces fluorescent compounds, which can be excited at wavelengths that are not commonly encountered in compounds present in real samples. For that, a whole cell-based sensing system for arsenite that employs cobA as the reporter gene was developed. The system utilizes genetically engineered bacteria that incorporate the specificity of the ars operon with the sensitivity of the cobA gene. The cobA gene codes for uroporphyrinogen III methyltransferase that converts the substrate uroporphyrinogen (urogen) III into two fluorescent compounds sirohydrochlorin and trimethylpyrrocorphin. Urogen III is ubiquitous within the cell, however, because the cells use it for vitamin B12 and siroheme biosynthesis, this sensing system is limited by substrate availability. By supplementing the media with ALA, a precursor of urogen III, a more stable and reproducible response was obtained. We observed three excitation maxima at 357, 378, and 498 nm, with a single emission maximum at 605 nm. Excitation at 498 nm was selected because it results in less background interference as most endogenous substances are not active at this wavelength. Advantages and limitations of using the cobA gene in whole-cell sensing applications are presented.

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“…In an attempt to understand how these factors impact bioremediation, reporter organisms have been developed that allow monitoring of microbial interaction with organic compounds in dynamic systems (6,20). Reporter organisms are genetically engineered organisms in which a reporter gene(s), such as lux, luc, or gfp, that encodes a detectable gene product is under regulatory control of an inducible catabolic operon (12). In general, due to a relatively short half-life, luciferase (lux or luc) is preferable in applications where dynamic measurement of gene expression is required (3,5,12,21).…”

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confidence: 99%

“…Reporter organisms are genetically engineered organisms in which a reporter gene(s), such as lux, luc, or gfp, that encodes a detectable gene product is under regulatory control of an inducible catabolic operon (12). In general, due to a relatively short half-life, luciferase (lux or luc) is preferable in applications where dynamic measurement of gene expression is required (3,5,12,21). However, several groups have observed that bioluminescence is sensitive to physiological and environmental factors (2,4,8,10,15,17,19,22).…”

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confidence: 99%

Effect of Temperature, pH, and Initial Cell Number on luxCDABE and nah Gene Expression during Naphthalene and Salicylate Catabolism in the Bioreporter Organism Pseudomonas putida RB1353

Dorn

Frye

Maier

2003

Appl Environ Microbiol

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One limitation of employing lux bioreporters to monitor in situ microbial gene expression in dynamic, laboratory-scale systems is the confounding variability in the luminescent responses. For example, despite careful control of oxygen tension, growth stage, and cell number, luminescence from Pseudomonas putida RB1353, a naphthalene-degrading lux bioreporter, varied by more than sevenfold during saturated flow column experiments in our laboratory. Therefore, this study was conducted to determine what additional factors influence the luminescent response. Specifically, this study investigated the impact of temperature, pH, and initial cell number (variations within an order of magnitude) on the peak luminescence of P. putida RB1353 and the maximum degradation rate (V max ) during salicylate and naphthalene catabolism. Statistical analyses based on general linear models indicated that under constant oxygen tension, temperature and pH accounted for 98.1% of the variability in luminescence during salicylate catabolism and 94.2 and 49.5% of the variability in V max during salicylate and naphthalene catabolism, respectively. Temperature, pH, and initial substrate concentration accounted for 99.9% of the variability in luminescence during naphthalene catabolism. Initial cell number, within an order of magnitude, did not have a significant influence on either peak luminescence or V max during salicylate and naphthalene catabolism. Over the ranges of temperature and pH evaluated, peak luminescence varied by more than 4 orders of magnitude. The minimum parameter deviation required to alter lux gene expression during salicylate and naphthalene catabolism was a change in temperature of 1°C, a change in pH of 0.2, or a change in initial cell number of 1 order of magnitude. Results from this study indicate that there is a need for careful characterization of the impact of environmental conditions on both the expression of the reporter and catabolic genes and the activities of the gene products. For example, even though lux gene expression was occurring at ϳ35°C, the luciferase enzyme was inactive. Furthermore, this study demonstrates that with careful characterization and standardization of measurement conditions, the attainment of a reproducible luminescent response and an understanding of the response are feasible.One of the major constraints on implementing in situ bioremediation is the lack of understanding of how physical, biological, and chemical factors affect microbial activity (7). In an attempt to understand how these factors impact bioremediation, reporter organisms have been developed that allow monitoring of microbial interaction with organic compounds in dynamic systems (6,20). Reporter organisms are genetically engineered organisms in which a reporter gene(s), such as lux, luc, or gfp, that encodes a detectable gene product is under regulatory control of an inducible catabolic operon (12). In general, due to a relatively short half-life, luciferase (lux or luc) is preferable in applications where dynamic measure...

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Peer Reviewed: Applications of Reporter Genes

Cited by 69 publications

References 28 publications

Purification and Characterization of Extracellular β-Galactosidase Secreted by Supension Cultured Rice (Oryza sativaL.) Cells

Purification and Characterization of Extracellular β-Galactosidase Secreted by Supension Cultured Rice (Oryza sativaL.) Cells

Novel reporter gene in a fluorescent‐based whole cell sensing system

Effect of Temperature, pH, and Initial Cell Number on luxCDABE and nah Gene Expression during Naphthalene and Salicylate Catabolism in the Bioreporter Organism Pseudomonas putida RB1353

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