Green Fluorescent Protein Emission Research Articles

Application of an Escherichia coli green fluorescent protein - based lysine biosensor under nonsterile conditions and autofluorescence background

To examine the utility of an Escherichia coli green fluorescent protein (GFP) containing biosensor for quantification of bioavailable lysine in selected feed samples under nonsterile conditions and to estimate the background fluorescence of analyzed feed samples and evaluate the risk of confounding GFP emission from the lysine assay organism. Escherichia coli lysine auxotroph GFP based biosensor was used to determine the percentage of bioavailable lysine in two samples of soybean-, cottonseed-, and meat and bone meal under nonsterile conditions. The fluorescence emitted by GFP was successfully measured using a spectrofluorimeter to monitor bacterial growth response to protein-derived lysine and lysine containing small peptides. The autofluorescence of analyzed feed samples at different concentrations could also be estimated. When feed protein concentrations are decreased, autofluorescence interference can be avoided. The E. coli lysine auxotroph GFP-based biosensor can successfully be used for the determination of bioavailable lysine in these selected animal feed proteins under nonsterile conditions. E. coli GFP biosensor for lysine has potential for routine application in animal feeds.

Directional Surface Plasmon‐Coupled Emission from a 3 nm Green Fluorescent Protein Monolayer

High-sensitivity detection schemes are of great interest for a number of applications. Unfortunately, such schemes are usually high-cost. We demonstrate a low-cost approach to a high-sensitivity detection scheme based on surface plasmon-coupled emission (SPCE). The SPCE of a monomolecular layer of green fluorescent protein (GFP) is reported here. The protein was electrostatically attached to a thin, SiO(2)-protected silver film deposited on a quartz substrate. The visible, directional emission of GFP was observed at a sharp, well-defined angle of 47.5 degrees from the normal to the coupling prism, and the spectrum corresponded to that of GFP. The SPCE resulting from the reverse Kretschmann configuration showed a 12-fold enhancement over the free space fluorescence. The directional emission was 97% p-polarized. The directionality and high polarization can be coupled with the intrinsic spectral resolution of SPCE to be used in the design miniaturized spectrofluorometers. The observation of SPCE in the visible region of the spectrum from a monolayer of protein opens up new possibilities in protein-based sensing.

Visualizing morphogenesis in transgenic zebrafish embryos using BODIPY TR methyl ester dye as a vital counterstain for GFP

Green fluorescent protein (GFP) technology is rapidly advancing the study of morphogenesis, by allowing researchers to specifically focus on a subset of labeled cells within the living embryo. However, when imaging GFP-labeled cells using confocal microscopy, it is often essential to simultaneously visualize all of the cells in the embryo using dual-channel fluorescence to provide an embryological context for the cells expressing GFP. Although various counterstains are available, part of their fluorescence overlaps with the GFP emission spectra, making it difficult to clearly identify the cells expressing GFP. In this study, we report that a new fluorophore, BODIPY TR methyl ester dye, serves as a versatile vital counterstain for visualizing the cellular dynamics of morphogenesis within living GFP transgenic zebrafish embryos. The fluorescence of this photostable synthetic dye is spectrally separate from GFP fluorescence, allowing dual-channel, three-dimensional (3D) and four-dimensional (4D) confocal image data sets of living specimens to be easily acquired. These image data sets can be rendered subsequently into uniquely informative 3D and 4D visualizations using computer-assisted visualization software. We discuss a variety of immediate and potential applications of BODIPY TR methyl ester dye as a vital visualization counterstain for GFP in transgenic zebrafish embryos.

Feasibility, sensitivity, and reliability of laser-induced fluorescence imaging of green fluorescent protein-expressing tumors in vivo

Whole-body imaging of green fluorescent protein (GFP) can be used to test the efficiency of gene carriers for in vivo transduction. The aim of the current study was to determine the sensitivity and the accuracy of a GFP imaging procedure by in vivo investigation of GFP-expressing tumor cells. An improved method of whole-body GFP imaging made use of a laser excitation source and band-pass filters matched specifically to GFP and constitutive tissue fluorescence emission bands. Processing of the primary GFP fluorescence images acquired by the CCD camera subtracted background tissue autofluorescence. Our approach achieved 100% sensitivity and specificity for in vivo detection of 10%-transfected BxPc3 pancreatic tumor after subcutaneous grafting or orthotopical implantation in the pancreas of nude mice. It also detected less transfected tumors (i.e., 1 to 5%) but with a loss in sensitivity (50% of cases). The system was employed over a 5-week period to monitor the persistence of GFP expression in 10%-transfected BxPc3 tumors orthotopically implanted in the pancreas of two nude mice, allowing the direct visualization of tumor progression and spread. In facilitating the temporal–spatial follow-up of GFP expression in vivo, the optimized laser-induced fluorescence imaging device can support preclinical investigations of vectors for therapeutic gene transduction through regular, harmless, real-time monitoring of theirin vivo transductional efficacy and persistence.

Green‐fluorescent proteins in Caribbean corals

Fluorescent pigments in several Indo‐Pacific and Caribbean anthozoans have recently been identified as proteins related to the Green‐fluorescent protein (GFP) of the hydromedusa Aequorea victoria. Here we show that GFP is widely distributed in many Caribbean species. The fluorescence excitation and emission spectra for the pigment are similar to those reported elsewhere for coral and noncoral GFP and the fluorescence quantum yield is estimated to be 35%. Spectral and molecular characterization of the isolated protein clearly show it to be GFP, and laboratory and in situ fluorescence measurements and Western blot analysis show that it is widespread. Bathymetric studies of GFP content using Western blots for the ecologically important congeneric corals Montastraea faveolata and Montastraea cavernosa show that there is no significant correlation between depth and GFP concentration. Nucleotide sequence data of GFP from M. faveolata and M. cavernosa show 88.2% sequence homology with each other and 46.4% homology with A. victoria GFP, whereas the percent homology with A. victoria at the amino acid level was 31.1 and 28.4% for M. cavernosa and M. faveolata, respectively, and 82.7% with each other. Measurements of reflectance and of the excitation spectrum for chlorophyll fluorescence in GFP‐containing corals indicate that GFP absorption, emission, and reflection have negligible impact on the level of solar radiation reaching the zooxanthellae and therefore play no role in coral photosynthesis by either addition or removal of photons.

Examination of Salmonella gene expression in an infected mammalian host using the green fluorescent protein and two-colour flow cytometry.

Quantitative data on Salmonella gene expression in infected hosts are largely lacking because of technical problems. One attractive reporter, the green fluorescent protein (GFP), is widely used in vitro but is difficult to quantify in infected tissues because of the preponderance of background particles with similar fluorescence. Here, bacterial GFP emission was spectrally distinguished from host autofluorescence by two-colour flow cytometry. Using this technique, the in vivo activity of three well-characterized promoters (PsicA, PssaH and PpagC) was determined. Their spatial and temporal activity patterns are in close agreement with predictions based on previous data and the colonization defects of corresponding deletion strains. To identify additional Salmonella promoters that are induced in infected animals, a genomic library was sorted by flow cytometry yielding four independent promoters. Genes expressed from PpibB and PsifA contribute to virulence, and chorismate mutase expressed from ParoQ might participate in aromatic acid biosynthesis, which is also required for virulence. Promoter P3g appears to be part of a mobile genetic element that is lacking in the completely sequenced strain LT2.

Stopwatch fluorescence: keeping time with DsRed

DsRed is a 28-kDa fluorescent protein responsible for the red colouration around the oral disk of a coral from the Discoma genus. Its recent rise to prominence has followed much effort to increase the fluorescent wavelength of the well-established Aequorea victoria green fluorescent protein (GFP). GFP variants with different emission colours are useful if the expression of numerous genes is to be followed simultaneously. DsRed has the longest reported emission maxima for a wild-type, spontaneously fluorescent protein (583 nm). However, it is hampered by a relatively low emission level and a parasitic green fluorescent peak that cross-talks with GFP emission. Verkhusha et al. 1 Verkhusha, V.V. et al. (2001) J. Biol. Chem. 10.1074/jbc.C100200200 Google Scholar overcame these problems by making a DsRed mutant that has lost the secondary fluorescence peak and that emits more fluorescence in cell culture than does the wild-type protein. The much slower maturation of DsRed compared with GFP was advantageous in studying expression over a period of time.

Elicitor action via cell membrane of a cultured rice cell demonstrated by the single-cell transient assay

In order to analyze intracellular signal transduction, we investigated the mechanism of chemical elicitor action by single-cell transient assay using green fluorescent protein (GFP) as a reporter gene. When the elicitor was applied from outside the cell into which the chitinase promoter and GFP reporter were introduced beforehand, fluorescence emission of GFP was observed. In contrast, when the elicitor was introduced in the cell to let the elicitor act from inside, no emission was observed. Addition of further elicitor from outside, however, did cause GFP emission. Therefore, it is clear that the elicitor does not act after entering the cell but that its signal is transduced into the cell via the cell membrane.

Localization and function of ferredoxin:NADP(+) reductase bound to the phycobilisomes of Synechocystis.

Each phycobilisome complex of the cyanobacterium Synechocystis PCC 6803 binds approximately 2.4 copies of ferredoxin:NADP(+) reductase (FNR). A mutant of this strain that carries an N-terminally truncated version of the petH gene, lacking the 9 kDa domain of FNR that is homologous to the phycocyanin-associated linker polypeptide CpcD, assembles phycobilisome complexes that do not contain FNR. Phycobilisome complexes, consisting of the allophycocyanin core and only the core-proximal phycocyanin hexamers from mutant R20, do contain a full complement of FNR. Therefore, the binding site of FNR in the phycobilisomes is not the core-distal binding site that is occupied by CpcD, but in the core-proximal phycocyanin hexamer. Phycobilisome complexes of a mutant expressing a fusion protein of the N-terminal domain of FNR and green fluorescent protein (GFP) contain this fusion protein in tightly bound form. Calculations of the fluorescence resonance energy transfer (FRET) characteristics between GFP and acceptors in the phycobilisome complex indicate that their donor-acceptor distance is between 3 and 7 nm. Fluorescence spectroscopy at 77K and measurements in intact cells of accumulated levels of P700(+) indicate that the presence of FNR in the phycobilisome complexes does not influence the distribution of excitation energy of phycobilisome-absorbed light between photosystem II and photosystem I, and also does not affect the occurrence of 'light-state transitions'.

Properties and Applications of EGFP, Enhanced Color Variants of GFP, and Unstable Derivatives of GFP

Abstract The green fluorescent protein (GFP) has great potential as a tool for biologists because it can be used as an in vivo real time reporter of protein localization and gene expression in a variety of experimental systems. Wild type GFP, however, has several undesirable properties including low brightness, a significant lag in the development of fluorescence, complex photoisomerization, inefficient protein folding at 37°C, and poor expression in several species. To improve upon these qualities, we have combined an ultra-bright variant of GFP, GFPmutl, with a synthetic gene sequence containing codons preferentially found in highly expressed human proteins. The combination of improved fluorescence intensity and higher expression levels yields an enhanced variant (EGFP) (Table 1) that greatly increases the sensitivity of this reporter. The natural green emission of GFP can conveniently be monitored by optics designed to detect fluorescein. There are, however, many potential applications for GFP that require additional emission colors.

Green fluorescent protein as a new expression marker in mycobacteria

This study describes the use and the advantages of the green fluorescent protein (GFP) as a reporter molecule for mycobacteria. The gfp gene from Aequorea victoria was placed under the control of the hsp60 promoter in the shuttle vector pGFM-11. The gfp expression in the recombinant Mycobacterium smegmatis and BCG was readily detected on agar plates by the development of an intense green fluorescence upon irradiation with long-wave u.v. light. In mycobacteria containing a pGFM-11 derivative that lacks the hsp60 promoter, no fluorescence was observed. However, this plasmid was successfully used as a promoter-probe vector to identify BCG promoters. The fluorescence emission of GFP in mycobacteria harbouring pGFM-11 and grown in liquid media could be quantified by spectrofluorimetry. This allowed for easy assessment of drug susceptibility. As GFP does not require the addition of substrates or co-factors, the green fluorescent bacilli could be directly observed within infected macrophages using fluorescence and laser confocal microscopy, or in tissue sections of infected mice. Finally, infected cells or free-living recombinant mycobacteria could also be analysed by flow cytometry. The GFP thus appears to be a convenient reporter for mycobacteria, allowing tracing of recombinant mycobacteria, isolation of promoters with interesting properties, in vivo drug testing and the development of new diagnostic tools.

Common Toxic Plant Ingestions

This article focuses on some of the more common dangerous plant and mushroom ingestions, discussing toxic components, early symptoms, and treatment. Among the plants considered are oleander, foxglove, hemlock, dieffenbachia, and Amanita phalloides. Details concerning recognition of toxic species are intentionally omitted in an effort to avoid potentially dangerous errors in identification.