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Green fluorescent protein-based biosensor to detect and quantify stress responses induced by DNA-degrading colicins

Green fluorescent protein-based biosensor to detect and quantify stress responses induced by DNA-degrading colicins

(2011) Green fluorescent protein-based biosensor to detect and quantify stress responses induced by DNA-degrading colicins.. Applied.[r]

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Detection of Enhanced Green Fluorescent Protein DNA in Pink Bollworm through Polymerase Chain Reaction

Detection of Enhanced Green Fluorescent Protein DNA in Pink Bollworm through Polymerase Chain Reaction

Fig.1. Digital photo of a 1.25% 0.5x TBE agarose gel with electrophoretically separated, PCR amplified DNA, showing two rows of 40 lanes on the same gel. Numbers refer to the first and last lane of each of four sets of 12 reactions plus 1 )))) g DNA standards (Gibco- BRL catalog no. 15628-050) in lanes 40 and 14 (lower row of wells). The 100 bp, and 600 bp bands of the DNA standards are labeled and appear brighter. Each set of 12 PCRs differed as follows: Lanes 1 to 12 contained PCRs with no template DNA. PCRs loaded in lanes 14 to 25 had 100 ng enhanced green fluorescent protein-negative pink bollworm template DNA. PCRs in lanes 27 to 39 contained 20 ng enhanced green fluorescent protein-positive pink bollworm template DNA. PCRs in lanes 1 to 12 lower row had 250 pg of enhanced green fluorescent protein- encoding plasmid as positive control template. Annealing temperatures in each set increased from left to right as follows: 50.6, 50.6, 51.6, 53.4, 55.8, 58.5, 61.4, 64.2, 66.7, 68.7, 69.7, 70.1 °C.
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Quantification and Improvement of Endotoxin Removal in BTEC's Downstream Process for Green Fluorescent Protein Production

Quantification and Improvement of Endotoxin Removal in BTEC's Downstream Process for Green Fluorescent Protein Production

Administration specifying limitations on the amount allowed in any injectable product. This topic is of interest to the Biomanufacturing Training and Education Center (BTEC) because E. coli is used in its process to express green fluorescent protein (GFP), which is used as a model biopharmaceutical in a number of courses. Several unit operations currently used to purify GFP such as chromatography and filtration are known to remove endotoxin. The purpose of this work was to determine the endotoxin concentration throughout the intermediate-scale downstream process at BTEC and to suggest possible process improvements to reduce endotoxin levels in the final product. The chromogenic kinetic Limulus Amebocyte Lysate assay was characterized and used for quantifying endotoxin. It was found that the first
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Multi state lasing in self assembled ring shaped green fluorescent protein microcavities

Multi state lasing in self assembled ring shaped green fluorescent protein microcavities

Despite increased interest in using biologically produced structures and materials for photonic applications, lasers have so far mostly relied on artificial materials. Recently, we have reported on a type of microlaser where the active component is formed by a live cell that is genetically programmed to pro- duce a highly fluorescent protein, the so-called eGFP (enhanced green fluorescent protein). 5,6 This work inspired further research on biologically produced gain materials in general; so far, the most notable results included the demon- stration of a silk fibroin based laser, 7 the development of microfluidic laser sensors based on fluorescent proteins, 8 and luciferins 9 as well as micro-droplet and distributed feedback lasers based on fluorescent flavin mononucleotides derived from the vitamin B2. 10,11 Whilst this research impressively illustrates the broad potential of biological materials for pro- viding optical gain, we believe that fluorescent proteins like eGFP retain a special position within the quickly growing family of biologically produced laser materials: First, nearly any organism can be genetically programmed to produce eGFP using straightforward genetic manipulation procedures, whereas many other biomaterials with attractive optical prop- erties are restricted to certain species. Second, fluorescent proteins are characterized by a unique molecular structure, comprising of a rigid protective nano-cylinder that is 2.4 nm in diameter and 4.2 nm in height and that encloses the much smaller light emitting fluorophore at its center (Fig. 1(a)). This so-called b-barrel structure maintains a defined distance between the fluorophores of neighbouring protein molecules. We have recently found that this separation prevents concen- tration induced quenching of the fluorescence in fluorescent
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Fluoresceination of Lactobacillus rhamnosus through the expression of green fluorescent protein

Fluoresceination of Lactobacillus rhamnosus through the expression of green fluorescent protein

We attempted to label a strain of L. rhamnosus with green fluorescent protein (GFP) as a tool to elucidate its mechanism of action. We produced pRNemgfp, carrying the gfpmut2 gene downstream of the erythromycin resistance gene, erm on pRN14. Culturing in MRS culture medium at 37°C produced L. rhamnosus with a green fluorescence in the exponential growth period. The pH regulation on the green fluorescence signal was indicated by artificial control of the pH in culture medium. By co-culturing GFP-labeled L. rhamnosus with mammalian cell lines, live L. rhamnosus was observed with GE1 and MC3T3-E1 until 4 h.Although we can successfully observed live L. rhamnosus in the mammalian culture, mammalian culture cells become fatigue to appear vacuoles in their cytoplasm, especially in GE1, by co-culturing them for 4h. We have to seek the best condition, such as bacterial dose, pH, and preculture medium, to add GFP-labeled L. rhamnosus into the culture medium of mammalian cells.
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Detection of chromosomes tagged with green fluorescent protein in live Arabidopsis thaliana plants

Detection of chromosomes tagged with green fluorescent protein in live Arabidopsis thaliana plants

So far, most in situ studies of gene positioning and changes in higher-order chromatin architecture have been carried out using FISH. This method can produce artifacts, however, because it requires the fixation and permeabilization of cells and denaturation of chromatin before hybridization with labeled single-strand probes [12]. It is also very difficult to perform kinetic studies with FISH to track the dynamics of chromatin movement in a single nucleus. Recent advances in the application of green fluorescent protein (GFP) as an in vivo tag of specific chromosomal regions promises to revolu- tionize our ability to observe chromatin-based processes in near real-time [13]. This technique, first established by Andrew Belmont and collaborators [14], entails the construc- tion of a fusion protein between GFP and the DNA-binding domain (DBD) of a known heterologous transcription factor. The binding site for the DBD is multimerized into a con- catameric array which is then inserted into the genome of animal or yeast cells. Expression of the GFP-DBD fusion protein results in fluorescent tagging of the concatamer in situ. This provides a ‘beacon’ that allows one to track the position of this region in the genome with high specificity and sensitivity. In yeast and animal cells, a single con- catamer insert can be visualized with high resolution and fidelity, and the application of this technique has provided new insights on chromosome behavior [15-17].
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Exciton dynamics in solid state green fluorescent protein

Exciton dynamics in solid state green fluorescent protein

We study the decay characteristics of Frenkel excitons in solid-state enhanced green fluorescent protein (eGFP) dried from solution. We further monitor the changes of the radiative exciton decay over time by crossing the phase transition from the solved to the solid state. Complex interactions between protonated and deproto- nated states in solid-state eGFP can be identified from temperature-dependent and time-resolved fluorescence experiments that further allow the determination of activation energies for each identified process.

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Incorporation of the Green Fluorescent Protein into the Herpes Simplex Virus Type 1 Capsid

Incorporation of the Green Fluorescent Protein into the Herpes Simplex Virus Type 1 Capsid

The herpes simplex virus type 1 (HSV-1) UL35 open reading frame (ORF) encodes a 12-kDa capsid protein designated VP26. VP26 is located on the outer surface of the capsid specifically on the tips of the hexons that constitute the capsid shell. The bioluminescent jellyfish (Aequorea victoria) green fluorescent protein (GFP) was fused in frame with the UL35 ORF to generate a VP26-GFP fusion protein. This fusion protein was fluorescent and localized to distinct regions within the nuclei of transfected cells following infection with wild-type virus. The VP26-GFP marker was introduced into the HSV-1 (KOS) genome resulting in recombinant plaques that were fluorescent. A virus, designated K26GFP, was isolated and purified and was shown to grow as well as the wild-type virus in cell culture. An analysis of the intranuclear capsids formed in K26GFP-infected cells revealed that the fusion protein was incorporated into A, B, and C capsids. Furthermore, the fusion protein incorporated into the virion particle was fluorescent as judged by fluorescence-activated cell sorter (FACS) analysis of infected cells in the absence of de novo protein synthesis. Cells infected with K26GFP exhibited a punctate nuclear fluorescence at early times in the replication cycle. At later times during infection a gener- alized cytoplasmic and nuclear fluorescence, including fluorescence at the cell membranes, was observed, con- firming visually that the fusion protein was incorporated into intranuclear capsids and mature virions.
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Dietary uptake of green fluorescent protein for delivery of dsRNA to induce RNA interference

Dietary uptake of green fluorescent protein for delivery of dsRNA to induce RNA interference

DNA encoding green fluorescent protein (gfp) was synthesised chemically and subcloned into plasmid pJepress607 (DNA.2.0, USA). Plasmid containing gfp was transformed into Escherichia coli JM109 cells, using pGEM-T Easy Vector Systems (Promega, Australia), according to the manufacturer’s instructions. Four colonies were selected for screening of recombinant plasmids. Colonies were seeded into 10 ml Luria Bertani (LB) medium supplemented with 100 mg/ ml hygromycin in a shaking incubator at 150 rpm overnight at 37°C. Recombinant plasmids were purified from bacteria using Wizard  Plus
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Live-Cell Analysis of a Green Fluorescent Protein-Tagged Herpes Simplex Virus Infection

Live-Cell Analysis of a Green Fluorescent Protein-Tagged Herpes Simplex Virus Infection

We have previously shown that a green fluorescent protein (GFP)-VP22 fusion protein (GFP-22) is competent for both intercellular movement and interaction with microtubules (8), suggesting that the addition of GFP onto the VP22 open read- ing frame has no effect on VP22 activities within the cell. In this report we describe the construction of an HSV-1 recom- binant in which we have exchanged the single copy of the VP22 open reading frame in the HSV-1 genome, gene UL49 (10), with a gene encoding GFP-22. Surprisingly, this virus is fully viable and exhibits growth kinetics similar to those of its pa- rental virus. Moreover, GFP-22 is incorporated into the virus particle with the same efficiency as VP22. The presence of GFP-22 in the virion results in fluorescent particles which are readily visualized with a light microscope. Furthermore, we show that newly synthesized GFP-22 is detectable as early as 3 h after infection at a high multiplicity, allowing the direct visualization of GFP-22 within live cells. As a consequence of such sensitive detection of GFP-22 throughout infection, we have been able to use time lapse confocal microscopy to mon- itor the trafficking of GFP-22 within individual cells, at both high and low multiplicities of infection, the results of which we present as time lapse animations. Thus, we have generated a reagent which will enable the visualization of several aspects of * Corresponding author. Mailing address: Marie Curie Research
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Silica Nanoparticles for Intracellular Protein Delivery: a Novel Synthesis Approach Using Green Fluorescent Protein

Silica Nanoparticles for Intracellular Protein Delivery: a Novel Synthesis Approach Using Green Fluorescent Protein

In this study, a novel approach for preparation of green fluorescent protein (GFP)-doped silica nanoparticles with a narrow size distribution is presented. GFP was chosen as a model protein due to its autofluorescence. Protein-doped nanoparticles have a high application potential in the field of intracellular protein delivery. In addition, fluorescently labelled particles can be used for bioimaging. The size of these protein-doped nanoparticles was adjusted from 15 to 35 nm using a multistep synthesis process, comprising the particle core synthesis followed by shell regrowth steps. GFP was selectively incorporated into the silica matrix of either the core or the shell or both by a one-pot reaction. The obtained nanoparticles were characterised by determination of particle size, hydrodynamic diameter, ζ -potential, fluorescence and quantum yield. The measurements showed that the fluorescence of GFP was maintained during particle synthesis. Cellular uptake experiments demonstrated that the GFP-doped nanoparticles can be used as stable and effective fluorescent probes. The study reveals the potential of the chosen approach for incorporation of functional biological macromolecules into silica nanoparticles, which opens novel application fields like intracellular protein delivery.
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Visualization of Intracellular Movement of Vaccinia Virus Virions Containing a Green Fluorescent Protein-B5R Membrane Protein Chimera

Visualization of Intracellular Movement of Vaccinia Virus Virions Containing a Green Fluorescent Protein-B5R Membrane Protein Chimera

We produced an infectious vaccinia virus that expressed the B5R envelope glycoprotein fused to the enhanced green fluorescent protein (GFP), allowing us to visualize intracellular virus movement in real time. Previous transfection studies indicated that fusion of GFP to the C-terminal cytoplasmic domain of B5R did not interfere with Golgi localization of the viral protein. To determine whether B5R-GFP was fully functional, we started with a B5R deletion mutant that made small plaques and inserted the B5R-GFP gene into the original B5R locus. The recombinant virus made normal-sized plaques and acquired the ability to form actin tails, indicating reversal of the mutant phenotype. Moreover, immunogold electron microscopy revealed that both intracellular enveloped virions (IEV) and extracellular enveloped virions contained B5R-GFP. By confocal microscopy of live infected cells, we visualized individual fluorescent particles, corresponding to IEV in size and shape, moving from a juxtanuclear location to the periphery of the cell, where they usually collected prior to association with actin tails. The fluorescent particles could be seen emanating from cells at the tips of microvilli. Using a digital camera attached to an inverted fluorescence microscope, we acquired images at 1 frame/s. At this resolution, IEV movement appeared saltatory; in some frames there was no net movement, whereas in others movement exceeded 2 ␮ m/s. Further studies indicated that IEV movement was reversibly arrested by the microtubule-depolymerizing drug nocodazole. This result, together with the direction, speed, and saltatory motion of IEV, was consistent with a role for microtubules in intracellular transport of IEV.
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A "humanized" green fluorescent protein cDNA adapted for high-level expression in mammalian cells.

A "humanized" green fluorescent protein cDNA adapted for high-level expression in mammalian cells.

The usefulness of the jellyfish gfp10 gene as a reporter in prokaryotes and animals (4) opened new perspectives in gene delivery technologies. Green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a protein of 238 amino acids which absorbs blue light (major peak at 395 nm) and emits green light (major peak at 509 nm) (27, 29, 36). The GFP hexapeptide chromophore starts at amino acid 64 and is de- rived from the primary amino acid sequence through the cy- clization of serine-dehydrotyrosine-glycine within this hexa- peptide (6, 32). The light-stimulated GFP fluorescence is species independent and does not require any cofactors, substrates, or additional gene products from A. victoria (4), thus allowing detection in living cells. The small size of gfp10 and the easy real-time detection of the product make it an ideal reporter for most viral vectors, AAV included. However, our initial attempt to show the expression of the jellyfish GFP reporter gene delivered into a human cell by a recombinant AAV (rAAV) was unsuccessful. We hypothesized that one of the reasons for the low expression of GFP was the poor translation efficiency of the mRNA in the human cell environment, which is char- acterized by a set of isoacceptor tRNAs that are different than
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The Role of Green Fluorescent Protein (GFP) in Transgenic Plants to Reduce Gene Silencing Phenomena

The Role of Green Fluorescent Protein (GFP) in Transgenic Plants to Reduce Gene Silencing Phenomena

Two plasmid vectors, pHVS and pHV, were constructed based on the pUC19 vector (Fig. 7 A and B). pHV contains the hygromycin phosphotransferase coding region, hpt (1.0kb), under regulatory control of the cauliflower mosaic virus (CaMV) 35S promoter, 35Spro, and the modified proglycinin (A1aB1b) cDNA, V3–1 (1.4kb), with a synthetic DNA encoding four continuous methionines. pHVS contains additionally a modified jellyfish green fluorescent protein coding region, sGFP(S65T) (0.8kb), under regulatory control of 35Spro in the flanking region of the V3–1 gene (El-Shemy et al,, 2004, 2006, 2007). Initiation and proliferation of embryogenic cultures Transformation and regeneration systems for soybean were optimized according to methods described elsewhere
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Green Fluorescent Protein Tagging Drosophila Proteins at Their Native Genomic Loci With Small P Elements

Green Fluorescent Protein Tagging Drosophila Proteins at Their Native Genomic Loci With Small P Elements

We describe a technique to tag Drosophila proteins with GFP at their native genomic loci. This technique uses a new, small P transposable element (the Wee-P) that is composed primarily of the green fluorescent protein (GFP) sequence flanked by consensus splice acceptor and splice donor sequences. We demonstrate that insertion of the Wee-P can generate GFP fusions with native proteins. We further demonstrate that GFP-tagged proteins have correct subcellular localization and can be expressed at near-normal levels. We have used the Wee-P to tag genes with a wide variety of functions, including transmembrane proteins. A genetic analysis of 12 representative fusion lines demonstrates that loss-of-function phenotypes are not caused by the Wee-P insertion. This technology allows the generation of GFP-tagged reagents on a genome- wide scale with diverse potential applications.
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Infection of Rrs1 barley by an incompatible race of the fungus Rhynchosporium secalis expressing the green fluorescent protein

Infection of Rrs1 barley by an incompatible race of the fungus Rhynchosporium secalis expressing the green fluorescent protein

Scald disease of barley, caused by the fungal pathogen Rhynchosporium secalis, is one of the most serious diseases of this crop worldwide. Disease control is achieved in part by deployment of major resistance (Rrs) genes in barley. However, in both susceptible and resistant barley plants, R. secalis is able to complete a symptomless infection cycle. To examine the R. secalis infection cycle, Agrobacterium tumefaciens-mediated transformation was used to generate R. secalis isolates expressing the green fluorescent protein or DsRed fluorescent protein, and that were virulent on an Rrs2 plant (cv. Atlas), but avirulent on an Rrs1 plant (cv. Atlas 46). Confocal laser scanning microscopy revealed that R. secalis infected the suscep- tible cultivar and formed an extensive hyphal network that followed the anticlinal cell walls of epidermal cells. In the resis- tant cultivar, hyphal development was more restricted and random in direction of growth. In contrast to earlier models of R. secalis infection, epidermal collapse was not observed until approximately 10 days post-inoculation in both cultivars. Sporulation of R. secalis was observed in both susceptible and resistant interactions. Observations made using the GFP- expressing isolate were complemented and confirmed using a combination of the fluorescent probes 5-chloromethylfluores- cein diacetate and propidium iodide, in the non-transformed wild-type isolate. The findings will enable the different Rrs genes to be better characterized in the effect they exert on pathogen growth and may aid in identification of the most effective resistance.
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Green Fluorescent Protein Reporter System with Transcriptional Sequence Heterogeneity for Monitoring the Interferon Response

Green Fluorescent Protein Reporter System with Transcriptional Sequence Heterogeneity for Monitoring the Interferon Response

Interferon (IFN) is produced and secreted by various mam- malian cell lines when they are infected by viruses, and it plays a regulatory role in innate immunity against viral infections. It also represents a significant therapeutic molecule in a number of viral diseases and cancers. IFN induces the Jak/STAT path- way leading to the activation and binding of transcriptional activators, e.g., the STAT/IRF9 complex, to the IFN-stimu- lated response element (ISRE) in the promoters of IFN-stim- ulated genes (2, 18). The transcription of IFN genes also is mediated via specific virus response elements (VREs) in the promoter; these sequences bind different IFN response factors (IRFs), such as IRF-3 and IRF-7, in the promoters of IFN genes (17). The VRE and ISRE sequences are found in IFN genes and IFN-stimulated genes; they partially overlap with each other, particularly the core AANNGAAA with the fol- lowing consensus: G(A)AAANNGAAAG/CT/C or A/GNG AAANNGAAACT (also in the complementary strand) (8, 19). Hundreds of virus- and IFN-stimulated genes exist in the hu- man genome (15), and although their promoters harbor spe- cific core sequence consensus elements, these sequences have context heterogeneity, variable reiterations, and distinct trans- activation potential. These sequence variations may account for responses to various types of viruses, IFNs, and IRFs. In this study, we have developed and validated a live cell-based enhanced green fluorescent protein (EGFP) reporter system employing 120 constructs containing multiple viruses and IFN response reporters that represent IFN system nucleic acid se- quence heterogeneity to monitor and assess promoter se- quence-function relationships during innate immunity. Several
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Hemagglutinin-Pseudotyped Green Fluorescent Protein-Expressing Influenza Viruses for the Detection of Influenza Virus Neutralizing Antibodies

Hemagglutinin-Pseudotyped Green Fluorescent Protein-Expressing Influenza Viruses for the Detection of Influenza Virus Neutralizing Antibodies

In order to determine whether GFP expression by these HA-pseudotyped influenza viruses could be used as an indica- tion of the neutralizing abilities of specific antibodies against HA, we investigated the neutralization capability of purified MAbs generated against A/BM/1/18 (Mount Sinai Hybridoma FIG. 2. Characterization of the HA-pseudotyped GFP-expressing influenza viruses. (A) Viral growth kinetics. Kinetics analyses of wild-type A/WSN/33 and HA-pseudotyped GFP-expressing viruses were performed in parental and HA-expressing MDCK cells. At indicated times postinfection, representative pictures were taken using fluorescent microscopy. At 72 h postinfection, light microscopy pictures were taken to show the cytopathic effect on the MDCK-HA-expressing cell lines. (B) Tissue culture supernatants shown in panel A were titrated by indirect immunofluorescence (wild-type [wt] WSN virus [top]) with a WSN polyclonal antibody or by GFP expression (HA-pseudotyped viruses [bottom]) on WSN-HA-expressing MDCK cells. hpi, hours postinfection. (C) Detection of NP and GFP. At 72 h postinfection, cell extracts were prepared and subjected to Western blotting for viral nucleoprotein (NP) and green fluorescent protein (GFP). The top two panels show Western blots of wild-type virus (WSN), while the bottom two panels show Western blots of WSN HA-pseudotyped GFP-expressing influenza virus (pWSN) in wild-type and HA-expressing MDCK cells. (D) Parental and HA-expressing MDCK cells were infected with the WSN-HA-pseudotyped GFP- expressing virus (MOI of 2). Twenty-four hours postinfection, representative pictures were taken using fluorescent microscopy. (E) Plaque assay. Confluent monolayers of the indicated MDCK cell lines were infected with the recombinant WSN-HA-pseudotyped GFP virus in the presence of agar media. Forty-eight hours postinfection, cells were stained with crystal violet.
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Linear correlation between average fluorescence intensity of green fluorescent protein and the multiplicity of infection of recombinant adenovirus

Linear correlation between average fluorescence intensity of green fluorescent protein and the multiplicity of infection of recombinant adenovirus

impacting transduced gene expression and protein produc- tion in these vectors have not been thoroughly investigated. Viral vector-derived protein expression is regulated by a number of factors, including the promoter used, gene copy number within the cell type transduced and the availability of cellular machinery for transcription and translation in host cells. Copy number of transduced gene is generally considered to be linearly correlated with the amount of target protein expressed. For viral vectors carrying the green fluorescent protein (GFP) the percentage of infection (POI) or mean fluorescence in- tensity (MFI) are considered to be linearly correlated with multiplicity of infection (MOI) under specific conditions [3-7]. However, how these correlations relate
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Green fluorescent protein as a marker for Pseudomonas spp.

Green fluorescent protein as a marker for Pseudomonas spp.

The development of sensitive methods for observing individual bacterial cells in a population in experimen- tal models and natural environments, such as in biofilms or on plant roots, is of great importance for studying these systems. We report the construction of plasmids which constitutively express a bright mutant of the green fluorescent protein of the jellyfish Aequorea victoria and are stably maintained in Pseudomonas spp. We demonstrate the utility of these plasmids to detect individual cells in two experimental laboratory systems: (i) the examination of a mixed bacterial population of Pseudomonas aeruginosa and Burkholderia cepacia attached to an abiotic surface and (ii) the association of Pseudomonas fluorescens WCS365 with tomato seedling roots. We also show that two plasmids, pSMC2 and pGB5, are particularly useful, because they are stable in the absence of antibiotic selection, they place an undetectable metabolic burden on cells that carry the plasmids, and cells carrying these constructs continue to fluoresce even after 7 days in culture without the addition of fresh nutrients. The construction of improved Escherichia coli-Pseudomonas shuttle vectors which carry mul- tiple drug resistance markers also is described.
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