Optimized expression of dual reporter genes in transient transfection of purifiedToxoplasma gondiiusing different promoters

2012 ◽  
Vol 58 (4) ◽  
pp. 483-489
Author(s):  
Yongxin Hao ◽  
Jianmin Yang ◽  
Xuelian Li ◽  
Jun Ding ◽  
Wei Zhang ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Reporter Genes ◽  
Transient Transfection ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Enhanced Yellow Fluorescent Protein ◽  
Dual Reporter ◽  
Green Fluorescent ◽  
Living Tissues

Fluorescent protein and luciferase genes are valuable reporter genes and have been widely used for noninvasive monitoring of gene expression in living tissues and cells. We tested expression of the dual reporter genes in transient transfection of purified Toxoplasma gondii tachyzoites. Two copies of the enhanced yellow fluorescent protein (EYFP) gene were put under the control of 3 representative T. gondii promoters (GRA1, SAG1, and DHFR). Fluorescence from each EYFP reporter was significantly higher than that from a green fluorescent protein (GFP) reporter. The GRA1-EYFP reporter gave the highest fluorescence. Although both fluorescence and luciferase were expressed in the dual reporter system, the luciferase reporter was more efficient than either the EYFP or GFP reporters, and it required fewer parasites to be successfully used.

scholarly journals Dual Reporter System for In Situ Detection of Plasmid Transfer under Aerobic and Anaerobic Conditions

2010 ◽  
Vol 76 (13) ◽  
pp. 4553-4556 ◽  
Author(s):  
Jaroslaw E. Król ◽  
Linda M. Rogers ◽  
Stephen M. Krone ◽  
Eva M. Top
Keyword(s):  
Fluorescent Protein ◽  
Plasmid Transfer ◽  
Reporter System ◽  
T7 Promoter ◽  
Dual Reporter ◽  
Green Fluorescent ◽  
In Situ Detection ◽  
Aerobic And Anaerobic ◽  
Rna Polymerase Gene

ABSTRACT We designed a new genetic tool to detect plasmid transfer under anaerobic and aerobic conditions. The system is based on the T7 RNA polymerase gene and a T7 promoter-driven oxygen-independent green fluorescent protein, evoglow, alone or in combination with red fluorescent protein DsRed. Constructs are available as plasmids and mini-mariner transposons.

scholarly journals Simultaneous Visualization of the Yellow and Green Forms of the Green Fluorescent Protein in Living Cells

1998 ◽  
Vol 46 (9) ◽  
pp. 1073-1076 ◽  
Author(s):  
Chris T. Baumann ◽  
Carol S. Lim ◽  
Gordon L. Hager
Keyword(s):  
Green Fluorescent Protein ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Simultaneous Detection ◽  
Argon Laser ◽  
Living Cells ◽  
Left Shoulder ◽  
Enhanced Yellow Fluorescent Protein ◽  
Green Fluorescent

In this study we sought to develop a method for the co-localization of proteins in living cells utilizing the enhanced green fluorescent protein (EGFP) and a redshifted EGFP variant, EYFP (enhanced yellow fluorescent protein). EYFP was expressed as an unsubstituted molecule while EGFP was fused to NF1 (EGFP-NF1), a transcription factor found exclusively in the nucleus. The Leica TCS SP laser scanning confocal microscope was used. This microscope allows the user to monitor the emitted light at defined wavelengths owing to the presence of a monochrometer in the emission light path. pEGFP-NF1 and pEYFP were co-expressed in the same cell and excited with the 476–nm and 488–nm argon laser lines. To separate the EYFP and EGFP fluorescence, EGFP-NF1 emission was recorded between 496 and 505 nm. These wavelengths are on the left shoulder of the EGFP emission peak and exclude most of the EYFP fluorescence. The EYFP emission was followed between 670 and 754 nm, utilizing the tail of EYFP emission that extends well beyond that for EGFP. Under these conditions we obtained excellent discrimination between EYFP fluorescence and EGFP-NF1 emission. These observations demonstrate that EYFP- and EGFP-substituted chimeras can be used for simultaneous detection in living cells.

scholarly journals Use of Green Fluorescent Protein Color Variants Expressed on Stable Broad-Host-Range Vectors to Visualize Rhizobia Interacting with Plants

2000 ◽  
Vol 13 (11) ◽  
pp. 1163-1169 ◽  
Author(s):  
Nico Stuurman ◽  
Cristina Pacios Bras ◽  
Helmi R. M. Schlaman ◽  
André H. M. Wijfjes ◽  
Guido Bloemberg ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Host Range ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Confocal Laser ◽  
Broad Host Range ◽  
Enhanced Yellow Fluorescent Protein ◽  
Green Fluorescent ◽  
Color Variants

We developed two sets of broad-host-range vectors that drive expression of the green fluorescent protein (GFP) or color variants thereof (henceforth collectively called autofluorescent proteins [AFPs]) from the lac promoter. These two sets are based on different replicons that are maintained in a stable fashion in Escherichia coli and rhizobia. Using specific filter sets or a dedicated confocal laser scanning microscope setup in which emitted light is split into its color components through a prism, we were able to unambiguously identify bacteria expressing enhanced cyan fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) in mixtures of the two. Clearly, these vectors will be valuable tools for competition, cohabitation, and rescue studies and will also allow the visualization of interactions between genetically marked bacteria in vivo. Here, we used these vectors to visualize the interaction between rhizobia and plants. Specifically, we found that progeny from different rhizobia can be found in the same nodule or even in the same infection thread. We also visualized movements of bacteroids within plant nodule cells.

scholarly journals Influence of the First Chromophore-Forming Residue on Photobleaching and Oxidative Photoconversion of EGFP and EYFP

2019 ◽  
Vol 20 (20) ◽  
pp. 5229 ◽  
Author(s):  
Tirthendu Sen ◽  
Anastasia Mamontova ◽  
Anastasia Titelmayer ◽  
Aleksander Shakhov ◽  
Artyom Astafiev ◽  
...  
Keyword(s):  
Excited State ◽  
Amino Acid ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Radiative Lifetime ◽  
Enhanced Yellow Fluorescent Protein ◽  
Atomistic Calculations ◽  
Oxidation Efficiency ◽  
Green Fluorescent

Enhanced green fluorescent protein (EGFP)—one of the most widely applied genetically encoded fluorescent probes—carries the threonine-tyrosine-glycine (TYG) chromophore. EGFP efficiently undergoes green-to-red oxidative photoconversion (“redding”) with electron acceptors. Enhanced yellow fluorescent protein (EYFP), a close EGFP homologue (five amino acid substitutions), has a glycine-tyrosine-glycine (GYG) chromophore and is much less susceptible to redding, requiring halide ions in addition to the oxidants. In this contribution we aim to clarify the role of the first chromophore-forming amino acid in photoinduced behavior of these fluorescent proteins. To that end, we compared photobleaching and redding kinetics of EGFP, EYFP, and their mutants with reciprocally substituted chromophore residues, EGFP-T65G and EYFP-G65T. Measurements showed that T65G mutation significantly increases EGFP photostability and inhibits its excited-state oxidation efficiency. Remarkably, while EYFP-G65T demonstrated highly increased spectral sensitivity to chloride, it is also able to undergo redding chloride-independently. Atomistic calculations reveal that the GYG chromophore has an increased flexibility, which facilitates radiationless relaxation leading to the reduced fluorescence quantum yield in the T65G mutant. The GYG chromophore also has larger oscillator strength as compared to TYG, which leads to a shorter radiative lifetime (i.e., a faster rate of fluorescence). The faster fluorescence rate partially compensates for the loss of quantum efficiency due to radiationless relaxation. The shorter excited-state lifetime of the GYG chromophore is responsible for its increased photostability and resistance to redding. In EYFP and EYFP-G65T, the chromophore is stabilized by π-stacking with Tyr203, which suppresses its twisting motions relative to EGFP.

scholarly journals Use of Fluorescent-Protein Tagging To Determine the Subcellular Localization of Mycoplasma pneumoniae Proteins Encoded by the Cytadherence Regulatory Locus

2004 ◽  
Vol 186 (20) ◽  
pp. 6944-6955 ◽  
Author(s):  
Tsuyoshi Kenri ◽  
Shintaro Seto ◽  
Atsuko Horino ◽  
Yuko Sasaki ◽  
Tsuguo Sasaki ◽  
...  
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Cytoskeletal Proteins ◽  
Host Cells ◽  
Protein Fusion ◽  
Enhanced Yellow Fluorescent Protein ◽  
Regulatory Locus ◽  
Green Fluorescent ◽  
Protein Tagging

ABSTRACT Mycoplasma pneumoniae lacks a cell wall but has internal cytoskeleton-like structures that are assumed to support the attachment organelle and asymmetric cell shape of this bacterium. To explore the fine details of the attachment organelle and the cytoskeleton-like structures, a fluorescent-protein tagging technique was applied to visualize the protein components of these structures. The focus was on the four proteins—P65, HMW2, P41, and P24—that are encoded in the crl operon (for “cytadherence regulatory locus”), which is known to be essential for the adherence of M. pneumoniae to host cells. When the P65 and HMW2 proteins were fused to enhanced yellow fluorescent protein (EYFP), a variant of green fluorescent protein, the fused proteins became localized at the attachment organelle, enabling visualization of the organelles of living cells by fluorescence microscopy. The leading end of gliding M. pneumoniae cells, expressing the EYFP-P65 fusion, was observed as a focus of fluorescence. On the other hand, when the P41 and P24 proteins were labeled with EYFP, the fluorescence signals of these proteins were observed at the proximal end of the attachment organelle. Coexpression of the P65 protein labeled with enhanced cyan fluorescent protein clearly showed that the sites of localization of P41 and P24 did not overlap that of P65. The localization of P41 and P24 suggested that they are also cytoskeletal proteins that function in the formation of unknown structures at the proximal end of the attachment organelle. The fluorescent-protein fusion technique may serve as a powerful tool for identifying components of cytoskeleton-like structures and the attachment organelle. It can also be used to analyze their assembly.

scholarly journals The Reporter System for GPCR Assay with the Fission YeastSchizosaccharomyces pombe

Scientifica ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Shintaro Sasuga ◽  
Toshiya Osada
Keyword(s):  
Fluorescent Protein ◽  
Signal To Noise Ratio ◽  
Biological Activities ◽  
Inducible Promoter ◽  
Upstream Region ◽  
Reporter Plasmid ◽  
Reporter System ◽  
High Background ◽  
Downstream Region ◽  
Green Fluorescent

G protein-coupled receptors (GPCRs) are associated with a great variety of biological activities. Yeasts are often utilized as a host for heterologous GPCR assay. We engineered the intense reporter plasmids for fission yeast to produce green fluorescent protein (GFP) through its endogenous GPCR pathway. As a control region of GFP expression on the reporter plasmid, we focused on seven endogenous genes specifically activated through the pathway. When upstream regions of these genes were used as an inducible promoter in combination with LPI terminator, themam2upstream region produced GFP most rapidly and intensely despite the high background. Subsequently, LPI terminator was replaced with the corresponding downstream regions. The SPBC4.01 downstream region enhanced the response with the low background. Furthermore, combining SPBC4.01 downstream region with the sxa2 upstream region, the signal to noise ratio was obviously better than those of other regions. We also evaluated the time- and dose-dependent GFP productions of the strains transformed with the reporter plasmids. Finally, we exhibited a model of simplified GPCR assay with the reporter plasmid by expressing endogenous GPCR under the control of the foreign promoter.

scholarly journals Genetic and transgenic reagents for Drosophila simulans, D. mauritiana, D. yakuba, D. santomea and D. virilis

2016 ◽  
Author(s):  
David L. Stern ◽  
Justin Crocker ◽  
Yun Ding ◽  
Nicolas Frankel ◽  
Gretchen Kappes ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Integration Site ◽  
Yellow Fluorescent Protein ◽  
Model Systems ◽  
Enhanced Yellow Fluorescent Protein ◽  
Functional Studies ◽  
Site Specific Integration ◽  
Genetic Strains ◽  
Fluorescent Molecules ◽  
Integration Efficiency

AbstractSpecies of the Drosophila melanogaster species subgroup, including the species D. simulans, D. mauritiana, D. yakuba, and D. santomea, have long served as model systems for studying evolution. Studies in these species have been limited, however, by a paucity of genetic and transgenic reagents. Here we describe a collection of transgenic and genetic strains generated to facilitate genetic studies within and between these species. We have generated many strains of each species containing mapped piggyBac transposons including an enhanced yellow fluorescent protein gene expressed in the eyes and a phiC31 attP site-specific integration site. We have tested a subset of these lines for integration efficiency and reporter gene expression levels. We have also generated a smaller collection of other lines expressing other genetically encoded fluorescent molecules in the eyes and a number of other transgenic reagents that will be useful for functional studies in these species. In addition, we have mapped the insertion locations of 58 transposable elements in D. virilis that will be useful for genetic mapping studies.

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