Use of the green fluorescent protein to locate α-amylase gene expression in barley grains

2002 ◽  
Vol 29 (9) ◽  
pp. 1037 ◽  
Author(s):  
Peter R. Matthews ◽  
Sarah Thornton ◽  
Frank Gubler ◽  
Rosemary White ◽  
John V. Jacobsen
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Confocal Laser ◽  
Aleurone Layer ◽  
Amylase Gene ◽  
Gfp Fluorescence ◽  
Amylase Production ◽  
Before And After ◽  
Barley Grains ◽  
Green Fluorescent

A green fluorescent protein (GFP) gene was cloned between the promoter and 3� regions from a barley high isoelectric point (pI) α-amylase gene, then inserted into barley. GFP fluorescence was used to locate and quantify expression of the transgene in barley grains following hydration. Light and confocal laser microscopy revealed fluorescence in the known regions of α-amylase synthesis in the scutellar epithelium, aleurone layer and embryonic axis. Fluorescence was quantified using a simple fluorescence assay, which showed induction of the transgene to mirror the induction of α-amylase in aleurone exposed to gibberellic acid. Expression from the transgene was also shown to be inhibited by abscisic acid, in the same way as expression of endogenous α-amylase genes. Overall, the transgenic grain revealed patterns of α-amylase expression before and after germination, and showed strong potential for further studies investigating both α-amylase production and transport of gibberellin in malting grain.

scholarly journals Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

2006 ◽  
Vol 14 (21) ◽  
pp. 9815 ◽  
Author(s):  
Alberto Diaspro ◽  
Silke Krol ◽  
Barbara Campanini ◽  
Fabio Cannone ◽  
Giuseppe Chirico
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Gfp Fluorescence ◽  
Green Fluorescent

Postharvest Handling Conditions Affect Internalization of Salmonella in Baby Spinach during Washing

2013 ◽  
Vol 76 (7) ◽  
pp. 1145-1151 ◽  
Author(s):  
VICENTE M. GÓMEZ-LÓPEZ ◽  
ALICIA MARÍN ◽  
ANA ALLENDE ◽  
LARRY R. BEUCHAT ◽  
MARÍA I. GIL
Keyword(s):  
Green Fluorescent Protein ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Negative Temperature ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Temperature Differential ◽  
Postharvest Handling ◽  
Green Fluorescent ◽  
Spinach Leaves

Internalization of foodborne pathogens in fruits and vegetables is an increasing safety concern. The aim of this research was to assess the potential for internalization of an enteric pathogen (Salmonella enterica serotype Typhimurium) in a leafy vegetable (baby spinach) during washing as influenced by three postharvest handling conditions: (i) illumination, (ii) negative temperature differential, and (iii) relative humidity (RH). To compare these potential postharvest handling conditions, leaves were exposed to different levels of illumination (0, 1,000, and 2,000 lx), temperature differential (5, 11, 14, 20, and 26uC), and RH (99, 85, and 74%) for a short time before or during washing. Washing of baby spinach was carried out in water containing green fluorescent protein–tagged Salmonella Typhimurium (6.5 log CFU/ml) at 5uC for 2 min, followed by surface disinfection with chlorine (10,000 μg/ml) for 1 min, two rinses in water for 10 s, and spin drying for 15 s. Internalization was assessed by enumerating the pathogen on Salmonella-Shigella agar and by confocal laser scanning microscopy. Illumination of spinach leaves before and during washing and a negative temperature differential during washing did not significantly (P > 0.05) increase the number of internalized bacteria. However, exposure of leaves to low-RH conditions before washing, which reduced the tissue water content, decreased internalization of Salmonella compared with internalization in baby spinach exposed to high RH (P ≤ 0.05). Green fluorescent protein–tagged Salmonella Typhimurium was visualized by confocal laser scanning microscopy at a depth of up to 30 μm beneath the surface of spinach leaves after exposure to a high inoculum level (8 log CFU/ml) for an extended time (2 h). Results show that internalization of Salmonella into baby spinach leaves can occur but can be minimized under specific postharvest handling conditions such as low RH.

Migration of Salmonella Enteritidis Phage Type 30 through Almond Hulls and Shells

2008 ◽  
Vol 71 (2) ◽  
pp. 397-401 ◽  
Author(s):  
MICHELLE D. DANYLUK ◽  
MARIA T. BRANDL ◽  
LINDA J. HARRIS
Keyword(s):  
Green Fluorescent Protein ◽  
Laser Scanning ◽  
Salmonella Enteritidis ◽  
Fluorescent Protein ◽  
Phage Type ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Aqueous Environment ◽  
Serovar Typhimurium ◽  
Green Fluorescent

The ability of Salmonella to migrate from an external aqueous environment through the almond hull and shell, and to colonize the kernel, was evaluated in two ways. First, the outer surface of shell halves from five varieties of almonds that differed in shell hardness were placed in contact with a suspension of Salmonella enterica serovar Enteritidis phage type 30 for 24hat24°C. Salmonella Enteritidis was isolated from the inside of these almond shells in 46 and 100% of the samples, by direct swabbing of the inner surface of the shell and by enrichment from the swab, respectively. These findings suggested that hardness of the shell is not a significant factor in the migration of the pathogen through that tissue. In addition, both motile and nonmotile strains of S. enterica serovar Typhimurium migrated through the almond shells to the same extent under the conditions of this assay, indicating that bacterial migration through the wet shell may be a passive process. Second, whole almonds (intact hull, shell, and kernel) were soaked for 24 to 72 h at 24°C in a suspension of Salmonella Enteritidis phage type 30 labeled with the green fluorescent protein. Green fluorescent protein–labeled Salmonella cells were observed on the outer and inner surfaces of both the almond hull and shell, and on the kernel, by confocal laser scanning microscopy. Our data provide direct evidence that wet conditions allow for Salmonella migration through the hull and shell and onto the almond kernel, thus providing a means by which almond kernels may become contaminated in the field.

scholarly journals Systemic Colonization of Potato Plants by a Soilborne, Green Fluorescent Protein-Tagged Strain of Dickeya sp. Biovar 3

2010 ◽  
Vol 100 (2) ◽  
pp. 134-142 ◽  
Author(s):  
Robert Czajkowski ◽  
Waldo J. de Boer ◽  
Henk Velvis ◽  
Jan M. van der Wolf
Keyword(s):  
Green Fluorescent Protein ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Agar Medium ◽  
Lateral Roots ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Potato Plants ◽  
Soil Inoculation ◽  
Green Fluorescent

Colonization of potato plants by soilborne, green fluorescent protein (GFP)-tagged Dickeya sp. IPO2254 was investigated by selective plating, epifluorescence stereo microscopy (ESM), and confocal laser scanning microscopy (CLSM). Replicated experiments were carried out in a greenhouse using plants with an intact root system and plants from which ca. 30% of the lateral roots was removed. One day after soil inoculation, adherence of the pathogen on the roots and the internal colonization of the plants were detected using ESM and CLSM of plant parts embedded in an agar medium. Fifteen days post-soil inoculation, Dickeya sp. was found on average inside 42% of the roots, 13% of the stems, and 13% of the stolons in plants with undamaged roots. At the same time-point, in plants with damaged roots, Dickeya sp. was found inside 50% of the roots, 25% of the stems, and 25% of the stolons. Thirty days postinoculation, some plants showed true blackleg symptoms. In roots, Dickeya sp. was detected in parenchyma cells of the cortex, both inter- and intracellularly. In stems, bacteria were found in xylem vessels and in protoxylem cells. Microscopical observations were confirmed by dilution spread-plating the plant extracts onto agar medium directly after harvest. The implications of infection from soilborne inoculum are discussed.

scholarly journals Green Fluorescent Protein as a Novel Indicator of Antimicrobial Susceptibility in Aureobasidium pullulans

2001 ◽  
Vol 67 (12) ◽  
pp. 5614-5620 ◽  
Author(s):  
Jeremy S. Webb ◽  
Sarah R. Barratt ◽  
Hristo Sabev ◽  
Marianne Nixon ◽  
Ian M. Eastwood ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Viability ◽  
Aureobasidium Pullulans ◽  
Fluorescent Protein ◽  
Fungal Species ◽  
Antimicrobial Compounds ◽  
Viable Cells ◽  
Gfp Fluorescence ◽  
Highly Correlated ◽  
Green Fluorescent

ABSTRACT Presently there is no method available that allows noninvasive and real-time monitoring of fungal susceptibility to antimicrobial compounds. The green fluorescent protein (GFP) of the jellyfishAequoria victoria was tested as a potential reporter molecule for this purpose. Aureobasidium pullulans was transformed to express cytosolic GFP using the vector pTEFEGFP (A. J. Vanden Wymelenberg, D. Cullen, R. N. Spear, B. Schoenike, and J. H. Andrews, BioTechniques 23:686–690, 1997). The transformed strain Ap1 gfp showed bright fluorescence that was amenable to quantification using fluorescence spectrophotometry. Fluorescence levels in Ap1 gfp blastospore suspensions were directly proportional to the number of viable cells determined by CFU plate counts (r 2 > 0.99). The relationship between cell viability and GFP fluorescence was investigated by adding a range of concentrations of each of the biocides sodium hypochlorite and 2-n-octylisothiozolin-3-one (OIT) to suspensions of Ap1gfp blastospores (pH 5 buffer). These biocides each caused a rapid (<25-min) loss of fluorescence of greater than 90% when used at concentrations of 150 μg of available chlorine ml−1 and 500 μg ml−1, respectively. Further, loss of GFP fluorescence from A. pullulanscells was highly correlated with a decrease in the number of viable cells (r 2 > 0.92). Losses of GFP fluorescence and cell viability were highly dependent on external pH; maximum losses of fluorescence and viability occurred at pH 4, while reduction of GFP fluorescence was absent at pH 8.0 and was associated with a lower reduction in viability. When A. pullulanswas attached to the surface of plasticized poly(vinylchloride) containing 500 ppm of OIT, fluorescence decreased more slowly than in cell suspensions, with >95% loss of fluorescence after 27 h. This technique should have broad applications in testing the susceptibility of A. pullulans and other fungal species to antimicrobial compounds.

Non-viral genetic transfection of rat Schwann cells with FuGENE HD© lipofection and AMAXA© nucleofection is feasible but impairs cell viability

2010 ◽  
Vol 6 (4) ◽  
pp. 225-230 ◽  
Author(s):  
Armin Kraus ◽  
Joachim Täger ◽  
Konrad Kohler ◽  
Max Haerle ◽  
Frank Werdin ◽  
...  
Keyword(s):  
Cell Culture ◽  
Green Fluorescent Protein ◽  
Cell Viability ◽  
Schwann Cells ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Cell Counting ◽  
Gfp Fluorescence ◽  
Green Fluorescent ◽  
Viral Transfection

Purpose:To determine transfection efficiency of FuGENE HD© lipofection and AMAXA© nucleofection on rat Schwann cells (SC).Methods:The ischiadic and median nerves of 6-8 week old Lewis rats were cultured in modified melanocyte-growth medium. SCs were genetically transfected with green fluorescent protein (GFP) as reporter gene using FuGENE HD© lipofection and AMAXA© nucleofection. Transfection rates were determined by visualization of GFP fluorescence under fluorescence microscopy and cell counting. Transfected cell to non-transfected cell relation was determined.Results:Purity of Schwann cell culture was 88% as determined by immunohistologic staining. Transfection rate of FuGENE HD© lipofection was 2%, transfection rate of AMAXA© nucleofection was 10%. With both methods, Schwann cells showed pronounced aggregation behavior which made them unfeasible for further cultivation. Settling of Schwann cells on laminin and poly-l-ornithine coated plates was compromised by either method.Conclusion:Non-viral transfection of rat SC with FuGENE HD© lipofection and AMAXA© nucleofection is basically possible with a higher transfection rate for nucleofection than for lipofection. As cell viability is compromised by either method however, viral transfection is to be considered if higher efficiency is required.

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

2001 ◽  
Vol 183 (23) ◽  
pp. 6752-6762 ◽  
Author(s):  
Johan H. J. Leveau ◽  
Steven E. Lindow
Keyword(s):  
Growth Rate ◽  
Green Fluorescent Protein ◽  
Green Fluorescent Protein Expression ◽  
Fluorescent Protein ◽  
Proteolytic Degradation ◽  
Bacterial Cells ◽  
Prior Testing ◽  
Gfp Fluorescence ◽  
Green Fluorescent ◽  
Best Fit

ABSTRACT 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-β-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 Michaelis-Menten 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-gfpfusions 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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