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Determination of zearalenone using a bioassay based on a green fluorescent protein (GFP) fusion. Gliocladium roseum strain carrying a fusion of zearalenone esterase with GFP was incubated with increasing concentrations of zearalenone. The fluorescence of GFP was measured in an iCycler real-time thermal cycler. Inset: the same data plotted on a semilogarithmic scale. Points are averages of 5 measurements with error bars of ± 1 standard deviation.
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A method based on mRNA-templated ligation of splice-junction anchored DNA probes followed by PCR amplification of the ligated product has been developed for multiplexed detection of mRNA splice variants with...
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... Furthermore, it does not require substrates or additional energy such as often is the case in bioluminescence . In addition, GFP-labeled cells can be used for flow cytometry analysis and quantitative analysis by PCR (Utermark and Karlovsky 2006). Disadvantages of GFP are that its structure and flourescence are dependent on pH and presence of oxygen (Heim et al. 1994). ...
Roots serve a multitude of functions in plants including anchorage, acquisition of nutrients and water, and production of
exudates with growth regulatory properties. The root–soil interface, or rhizosphere, is the site of greatest biological and
chemical activity within the soil matrix. Plant growth-promoting rhizobacteria (PGPR) are known to influence plant health
by controlling plant pathogens or via direct enhancement of plant development in the laboratory and in greenhouse experiments.
Unfortunately, however, results in the field have been less consistent. The colonization of roots by inoculated bacteria is
an important step in the interaction between beneficial bacteria and the host plant. However, colonization is a complex phenomenon
influenced by many biotic and abiotic parameters, some of which are only now apparent. Monitoring fate and metabolic activity
of microbial inoculants as well as their impact on rhizosphere and soil microbial communities are needed to guarantee safe
and reliable application, independent of whether they are genetically modified or not. The first and most crucial prerequisite
for effective use of PGPRs is that strain identity and activity are continuously confirmed. A combination of both classical
and molecular techniques must be perfected for more effective monitoring of inoculants strain (both genetically modified and
unmodified) after release into the soil. Recent developments in techniques for studying rhizobacterial communities and detection
and tracking systems of inoculated bacteria are important in future application and assessment of effectiveness and consistent
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KeywordsRhizosphere colonization-Rhizobacteria-Monitoring methods-Molecular techniques-GFP-PCR-Marker gene-Microscopy
... Additional advantages are that GFP due to its tertiary barrel structure is very stable and can be applied in many different species. In addition, GFP labelled cells can be used for flow cytometry analysis and quantitative analysis by PCR (Utermark and Karlovsky 2006). Disadvantages of GFP are that its structure and fluorescence is dependent on pH and the presence of oxygen (Heim et al. 1994). ...
Plant growth promoting rhizobacteria (PGPR) include bacteria that fix nitrogen (e.g., Rhizobiaceae, Herbaspirillum, Azoarcus), produce phytohormones (e.g., Azospirillum) and provide protection against fungal and/or bacterial pathogens (e.g., Pseudomonas, Bacillus, Streptomyces). Interactions between PGPR and plants can be divided into different steps which include initial attraction, attachment,
proliferation and colonization e.g., of roots, stem, leaves and flowers. At the genetic level the expression of many bacterial
genes are altered during these processes. In addition to the interaction with the plant, PGPR interact and compete with the
endogenous microflora, consisting of other bacteria, fungi and/or mycorrhizal fungi. In the case of biocontrol bacterial strains,
a direct interaction with the pathogen is often required to suppress the disease. Microscopic analyses of plant growth promoting
rhizobacteria (PGPR) in their natural environment and in specific during their interaction(s) with the host plant(s) and/or
their target organism(s) is essential for the elucidation of their functioning and the successful application of commercial
inoculants. With the discovery and development of auto fluorescent proteins (AFPs) as markers and the development of highly
sophisticated fluorescence microscopes such as confocal laser scanning microscopes, a new dimension has been created for studying
PGPR in their natural environment. This paper will give a short overview on available tools, the application of AFPs in PGPR
research and some future perspectives. Several recent reviews will give the reader an option for further reading (Bloemberg
and Lugtenberg 2004; Chalfie and Kain 2005; Larrainzar etal. 2005; Rediers etal. 2005; Bloemberg and Camacho 2006).
Bacterial plasmids and chromosomes widely contain toxin-antitoxin (TA) loci, which are implicated in stress response, growth regulation and even tolerance to antibiotics and environmental stress. Type I TA systems consist of a stable toxin-expressing mRNA, which is counteracted by an unstable RNA antitoxin. The Long Direct Repeat (LDR-) D locus, a type I TA system of Escherichia Coli (E. coli) K12, encodes a 35 amino acid toxic peptide, LdrD. Despite being characterized as a bacterial toxin, causing rapid killing and nucleoid condensation, little was known about its function and its mechanism of toxicity. Here, we show that LdrD specifically interacts with ribosomes which potentially blocks translation. Indeed, in vitro translation of LdrD-coding mRNA greatly reduces translation efficiency. The structure of LdrD in a hydrophobic environment, similar to the one found in the interior of ribosomes was determined by NMR spectroscopy in 100% trifluoroethanol solution. A single compact α-helix was found which would fit nicely into the ribosomal exit tunnel. Therefore, we conclude that rather than destroying bacterial membranes, LdrD exerts its toxic activity by inhibiting protein synthesis through binding to the ribosomes.
The green fluorescent protein (GFP) from jellyfish Aequorea victoria has been shown to possess a munber of desirable traits. For example, GFP in cells can generate a bright green fluorescence when the cells are excited by blue or UV light. GFP can be detected conveniently without the need of exogenous substrates, cell fixation and permealization. Morever, GFP has more stabillity to photobleaching, oxidation, reduction, acid, alkali and many other chemical reagents. As a universal reporter especially in living cells and tissues, GFP has been hailed as the live molecular probe and widely used in animal study, plant study, microbe study and other researches. The basic research of GFP and its application in drug screening, drug treatment effectiveness evaluation, bacterial drug resistance, and tumor cell multidrug resistance were mainly reviewed in this paper.
In the use of non-antibody proteins as affinity reagents, diversity has generally been derived from oligonucleotide-encoded random amino acids. Although specific binders of high-affinity have been selected from such libraries, random oligonuc- leotides often encode stop codons and amino acid combinations that affect protein folding. Recently it has been shown that specific antibody binding loops grafted into heterologous proteins can confer the specific antibody binding activity to the created chimeric protein. In this paper, we examine the use of such antibody binding loops as diversity ele- ments. We first show that we are able to graft a lysozyme-binding antibody loop into green fluores- cent protein (GFP), creating a fluorescent protein with lysozyme-binding activity. Subsequently we have developed a PCR method to harvest random binding loops from antibodies and insert them at predefined sites in any protein, using GFP as an example. The majority of such GFP chimeras remain fluorescent, indicating that binding loops do not disrupt folding. This method can be adapted to the creation of other nucleic acid libraries where divers- ity is flanked by regions of relative sequence conservation, and its availability sets the stage for the use of antibody loop libraries as diversity elements for selection experiments.
Modern application of insecticides belonging to different chemical families to boost agricultural productivity has led to
their accumulation in soils to levels that affect, directly and indirectly, soil enzyme activities and physiol-ogical characteristics
of nontarget soil microflora including plant growth-promoting rhizobacteria, and, consequently the performance of crop plants.
Various biological strategies can be applied for removing toxic substances, including insecticides, from the environment and
are collectively known as bioremediation. Among biological approaches, the use of microbes with degradative ability is considered
the most efficient and cost-effective option to clean pesticide-contaminated sites. The present review focuses on the role
of naturally occurring rhizosphere microbes involved in degradation or transformation of insecticides.
Key Wordstransgene–rat–fluorescent protein–immunocytochemistry–Western blot–somatostatin–pituitary–cortex–striatum
In the use of non-antibody proteins as affinity reagents, diversity has generally been derived from oligonucleotide-encoded
random amino acids. Although specific binders of high-affinity have been selected from such libraries, random oligonucleotides
often encode stop codons and amino acid combinations that affect protein folding. Recently it has been shown that specific
antibody binding loops grafted into heterologous proteins can confer the specific antibody binding activity to the created
chimeric protein. In this paper, we examine the use of such antibody binding loops as diversity elements. We first show that
we are able to graft a lysozyme-binding antibody loop into green fluorescent protein (GFP), creating a fluorescent protein
with lysozyme-binding activity. Subsequently we have developed a PCR method to harvest random binding loops from antibodies
and insert them at predefined sites in any protein, using GFP as an example. The majority of such GFP chimeras remain fluorescent,
indicating that binding loops do not disrupt folding. This method can be adapted to the creation of other nucleic acid libraries
where diversity is flanked by regions of relative sequence conservation, and its availability sets the stage for the use of
antibody loop libraries as diversity elements for selection experiments.
