Green Fluorescent Protein as a Quantitative Reporter of Relative Promoter Activity in
            <i>E. coli</i>

Lissemore, James L.; Jankowski, Jakub; Thomas, C.; Mascotti, David P.; deHaseth, Pieter L.

doi:10.2144/00281st02

Cited by 34 publications

(25 citation statements)

References 30 publications

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“…Although the potential application of GFP as a reporter gene has been suggested (1,(9)(10)(11)(12) sient and stable expression systems. Their use as dynamic and semiquantitative reporters to monitor stimulus-induced promoter activity in mammalian cells has been exploited sparsely (4,(13)(14)(15).…”

Section: Discussionmentioning

confidence: 99%

Online monitoring of stimulus-induced gene expression in pancreatic beta-cells.

et al. 2001

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T he use of fluorescent proteins as a "protein-tag" in cell biology sheds new light on the mechanisms that determine key processes in cell function, such as subcellular localization of proteins, colocalization of proteins, physical interaction of proteins, and protein translocation. Used in a rational experimental set-up and in combination with techniques that allow high resolution and/or high dynamics of the obtained images, fluorescent proteins provide a tool that makes the phrase "seeing is believing" a reality in cell physiology. The generation of different colored members of the green fluorescent protein (GFP) family (1), i.e., blue (BFP, EBFP), cyan (ECFP), green (GFP, EGFP), and yellow (EYFP), as well as the cloning of new fluorescent proteins, such as the red fluorescent protein DsRed from Discosoma sp.(2), permitted fluorescent probes exhibiting distinct spectra of light/energy excitation and emission. This allowed on the one hand the use of a combination of different colors to monitor distinct proteins at the same time; on the other hand, it made it possible to develop tailored molecular probes to monitor protein interactions, substrate concentrations, enzyme activities, etc., at the subcellular level, partially based on fluorescence resonance energy transfer. A timely review on the potential application of fluorescent proteins based on the GFP family was published by Tsien (1).The use of fluorescent proteins as reporter genes, although proposed as genetic markers from the beginning by Chalfie et al. (3), has been scarce, except as a marker in transfection experiments, despite their potential for use as dynamic "reporters" compared with classical reporter genes.Here we show that fluorescent proteins, i.e., GFP S65T and DsRed, can serve as semiquantitative and dynamic reporters for stimulus-induced gene expression in general and in the context of stimulus-induced cell type-specific gene expression, exemplified in the insulin-producing pancreatic ␤-cell. Moreover, we show that fluorescent proteins do not only allow monitoring of stimulus-induced gene expression at the single-cell level but also at the level of a micro-organ-here, the islet of Langerhans. Finally, use of fluorescent proteins with discrete excitation/emission properties, i.e., GFP S65T and DsRed, allows the simultaneous monitoring of two genes in the same cell, here exemplified by the stimulus-induced rat insulin 1 (rIns1) promoter-driven DsRed expression and the rat ␤-cell-active glucokinase (r␤GK) gene promoter-driven GFP expression. RESEARCH DESIGN AND METHODSExpression vectors. The construction of prIns1.GFP and pCMV.GFP was described elsewhere (4). Adenovirus (Ad)-rIns1.GFP (5) was provided by Dr. Lina Moitoso de Vargas (New England Medical School, Boston, MA). Plasmid prIns1.DsRed was generated by exchanging the GFP S65T -bGHpA cassette with the DsRed-SV40pA cassette from pDsRed1-1 (Clontech, Palo Alto, CA). Plasmid pr␤GK.GFP was constructed by exchanging the CAT-SV40pA cassette in pr␤GK-278.CAT (6) with the GFP S65T -bGHpA cassette...

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Section: Discussionmentioning

confidence: 99%

Online monitoring of stimulus-induced gene expression in pancreatic beta-cells.

et al. 2001

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“…al. (32) describe methods for measuring GFP expression in E. coli cells grown on soft agar. Scholz et.…”

Section: Introductionmentioning

confidence: 99%

Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells

2005

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Green fluorescent protein (GFP) has gained widespread use as a tool to visualize spatial and temporal patterns of gene expression in vivo. However, it is not generally accepted that GFP can also be used as a quantitative reporter of gene expression. We report that GFP is a reliable reporter of gene expression in individual eukaryotic cells when fluorescence is measured by flow cytometry. Two pieces of evidence that support this conclusion are that 1. GFP fluorescence increases in direct proportion to the GFP gene copy number delivered to cells by a replication-defective adenovirus vector, Ad.CMV-GFP, and that 2. the intensity of GFP fluorescence is directly proportional to GFP mRNA abundance in cells. This conclusion is further supported by the fact that the induction of GFP gene expression from two inducible promoters (i.e., the TRE and ICP0 promoters) is readily detected by flow cytometric measurement of GFP fluorescent intensity. Collectively, the results presented herein indicate that GFP fluorescence is a reliable and quantitative reporter of underlying differences in gene expression.

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“…GFP fluorescence has been validated as a reporter output by direct comparison to more traditional reporter proteins such as ␤-galactosidase (28,39) and chloramphenicol acetyltransferase (1). Still, two properties that are unique to GFP have been recognized as less desirable when trying to infer promoter activity from fluorescence measurements.…”

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confidence: 99%

Predictive and Interpretive Simulation of Green Fluorescent Protein Expression in Reporter Bacteria

2001

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We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P A1/O4/O3 in response to increasing concentrations of the inducer IPTG (isopropyl-␤-D-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P A1/O4/O3 . Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP[LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by MichaelisMenten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in Erwinia herbicola than in Escherichia coli. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-gfp fusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP fluorescence data that are obtained with that reporter.

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Green Fluorescent Protein as a Quantitative Reporter of Relative Promoter Activity in E. coli

Cited by 34 publications

References 30 publications

Online monitoring of stimulus-induced gene expression in pancreatic beta-cells.

Online monitoring of stimulus-induced gene expression in pancreatic beta-cells.

Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells

Predictive and Interpretive Simulation of Green Fluorescent Protein Expression in Reporter Bacteria

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