Academic literature on the topic 'EGFP (enhanced green fluorescent protein)'
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Journal articles on the topic "EGFP (enhanced green fluorescent protein)"
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Baumann, Chris T., Carol S. Lim, and Gordon L. Hager. "Simultaneous Visualization of the Yellow and Green Forms of the Green Fluorescent Protein in Living Cells." Journal of Histochemistry & Cytochemistry 46, no. 9 (September 1998): 1073–76. http://dx.doi.org/10.1177/002215549804600911.
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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.
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Takenaka-Uema, Akiko, Yousuke Murata, Fumihiro Gen, Yukari Ishihara-Saeki, Ken-ichi Watanabe, Kazuyuki Uchida, Kentaro Kato, et al. "Generation of a Recombinant Akabane Virus Expressing Enhanced Green Fluorescent Protein." Journal of Virology 89, no. 18 (July 8, 2015): 9477–84. http://dx.doi.org/10.1128/jvi.00681-15.
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ABSTRACTWe generated a recombinant Akabane virus (AKAV) expressing enhanced green fluorescence protein (eGFP-AKAV) by using reverse genetics. We artificially constructed an ambisense AKAV S genome encoding N/NSs on the negative-sense strand, and eGFP on the positive-sense strand with an intergenic region (IGR) derived from the Rift Valley fever virus (RVFV) S genome. The recombinant virus exhibited eGFP fluorescence and had a cytopathic effect in cell cultures, even after several passages. These results indicate that the gene encoding eGFP in the ambisense RNA could be stably maintained. Transcription of N/NSs and eGFP mRNAs of eGFP-AKAV was terminated within the IGR. The mechanism responsible for this appears to be different from that in RVFV, where the termination sites for N and NSs are determined by a defined signal sequence. We inoculated suckling mice intraperitoneally with eGFP-AKAV, which resulted in neurological signs and lethality equivalent to those seen for the parent AKAV. Fluorescence from eGFP in frozen brain slices from the eGFP-AKAV-infected mice was localized to the cerebellum, pons, and medulla oblongata. Our approach to producing a fluorescent virus, using an ambisense genome, helped obtain eGFP-AKAV, a fluorescent bunyavirus whose viral genes are intact and which can be easily visualized.IMPORTANCEAKAV is the etiological agent of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic loss to the livestock industry. We successfully generated a recombinant enhanced green fluorescent protein-tagged AKAV containing an artificial ambisense S genome. This virus could become a useful tool for analyzing AKAV pathogenesis in host animals. In addition, our approach of using an ambisense genome to generate an orthobunyavirus stably expressing a foreign gene could contribute to establishing alternative vaccine strategies, such as bivalent vaccine virus constructs, for veterinary use against infectious diseases.
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Tandio Saputro, Shania Safera, Khayu Wahyunita, Astutiati Nurhasanah, Yudhi Nugraha, Irvan Faizal, Sabar Pambudi, and Andri Pramesyanti Pramono. "Expression of modified enhanced green fluorescent polyarginine protein in Saccharomyces cerevisiae INVSc1." F1000Research 12 (January 3, 2023): 1. http://dx.doi.org/10.12688/f1000research.123181.1.
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Background: The enhanced green fluorescent protein (EGFP) gene is a reporter gene that can be used to optimize protein isolation procedures and the functional working of a transduction protein. EGFP, with the addition of eleven arginine residues, has been engineered to functionally improve the protein transduction process, which can later be used for cell reprogramming like induced pluripotent stem cells. The addition of six histidine amino acid residues at its C-terminal is intended for the protein isolation process using the His-tag antibody. Methods: The study aimed to investigate the optimization of the EGFP polyarginine protein expression in Saccharomyces cerevisiae in sufficient quantities for the protein isolation stage. This study also analyzed EGFP expression without polyarginine to analyze the polyarginine addition effect on expression processes. Protein expression was qualitatively measured by looking at expression fluorescence and protein levels of EGFP and EGFP - PolyR proteins. Results: Bands on Western Blots with 6×His-tag monoclonal antibody (primary antibody) and Goat anti-mouse IgG HRP (secondary antibody) showed the EGFP polyarginine and EGFP proteins were expressed in Saccharomyces cerevisiae INVSc1 at relatively low levels. The lyticase incubation time modification and administration of 3-5 kDa microfilter to concentrate increased the yield of isolated protein. Conclusions: The sufficient amount of protein isolation in S. cerevisiae can be achieved by using lyticase and sonicators combination for the lysis process.
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Mi, H. W., M. C. Lee, Y. C. Chiang, L. P. Chow, and C. P. Lin. "Single-Molecule Imaging of Bmp4 Dimerization on Human Periodontal Ligament Cells." Journal of Dental Research 90, no. 11 (August 12, 2011): 1318–24. http://dx.doi.org/10.1177/0022034511418340.
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We expressed bone morphogenetic protein 4 (BMP4) fused with enhanced green fluorescent protein (BMP4-EGFP) in the secretory pathways of producer cells. Fluorescent EGFP was acquired only after we interrupted the transport of BMP4-EGFP by culturing cells at a lower temperature (20°C), and the dynamics of BMP4-EGFP could be monitored by single-molecule microscopy. Western blotting analysis confirmed that exposure to low temperature helped the integrated formation of BMP4-EGFP fusion proteins. In this study, for the first time, we could image the fluorescently labeled BMP4 molecules localized on the plasma membrane of living hPDL cells. The one-step photobleaching with EGFP and the “blinking” behavior of quantum dots suggest that the fluorescent spots represent the events of single BMP4 molecules. Single-molecule tracking showed that the BMP receptors (BMPR) dimerize after BMP4 stimulation, or that a complex of one BMP4 molecule and a pre-formed BMPR dimer develops first, followed by the binding of the second BMP4 molecule. Furthermore, BMP4-EGFP enhanced the osteogenic differentiation of hPDL cells via signal transduction involving BMP receptors. This single-molecule imaging technique might be a valuable tool for the future development of BMP4 gene therapy and regenerative medicine mediated by hPDLs. Abbreviations: BMP4, bone morphogenetic protein 4; BMPR, BMP receptor; EGFP, enhanced green fluorescent protein; hPDL cells, human periodontal ligament cells; QDs, quantum dots; TIRFM, total internal reflection fluorescence microscopy; 293 cells, human embryonic kidney cells; oDM, osteogenic differentiation medium; HcoI, type I collagen; ALP, alkaline phosphatase; BSP, bone sialoprotein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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Mizozoe, Otaki, and Aikawa. "The Mechanism of Chlorine Damage Using Enhanced Green Fluorescent Protein-Expressing Escherichia coli." Water 11, no. 10 (October 16, 2019): 2156. http://dx.doi.org/10.3390/w11102156.
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This study investigated how chlorine inactivates and damages Escherichia coli cells. E. coli that had transformed to express enhanced green fluorescent protein (EGFP) at the cytoplasm was treated with chlorine. Damage to the cell membrane and cell wall was analyzed by measuring the fluorescence intensity of the leaked EGFP, then accounting for the fluorescence deterioration. At pH 7, E. coli was lethally damaged after treatment with chlorine, but significant leakage of EGFP was not observed. In contrast, significant leakage of EGFP was observed at pH 9, even though E. coli was not as inactivated as it was at pH 7. Flow cytometry was used to confirm the fluorescence intensity of the remaining EGFP inside the cells. No significant fluorescence loss was observed in the cells at pH 7. However, at pH 9, the fluorescence intensity in the cells decreased, indicating leakage of EGFP. These results suggest that hypochlorous acid inactivates E. coli without damaging its cell membrane and cell wall, whereas the hypochlorite ion inactivates E. coli by damaging its cell membrane and cell wall. It was possible to confirm the chlorine damage mechanism on E. coli by measuring the fluorescence intensity of the leaked EGFP.
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dos Santos, Nathalia Vieira, Carolina Falaschi Saponi, Tamar Louise Greaves, and Jorge Fernando Brandão Pereira. "Revealing a new fluorescence peak of the enhanced green fluorescent protein using three-dimensional fluorescence spectroscopy." RSC Advances 9, no. 40 (2019): 22853–58. http://dx.doi.org/10.1039/c9ra02567g.
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Krasowska, Joanna, Monika Olasek, Agnieszka Bzowska, Patricia L. Clark, and Beata Wielgus-Kutrowska. "The comparison of aggregation and folding of enhanced green fluorescent protein (EGFP) by spectroscopic studies." Spectroscopy 24, no. 3-4 (2010): 343–48. http://dx.doi.org/10.1155/2010/186903.
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GFP (Green Fluorescent Protein) is well known for its unique chromophore which is formed by autocatalytic cyclization of a polypeptide backbone of Ser65, Tyr66 and Gly67, and is able to emit green visible light. Due to unusual chromophore responsible for the fluorescence GFP and its mutants (e.g., EGFP) have become widely used reporter proteins in molecular biology and biotechnology. GFP can easily be fused to any protein of interest and co-expressed in cells; the GFP fluorescence is then used to visualize the distribution, transport and aggregation of the protein in the cell. However, GFP has a tendency to aggregate itself, and also formation of its chromophore critically depends on the presence of reducing agents. Therefore we have undertaken spectroscopic kinetic studies of EGFP folding and aggregation as a function of pH, and in the presence of various reducing agents, to study the competition between these two processes. The best conditions for folding of EGFP provides BME as a reducing agent. Aggregation of EGFP depends strongly on pH, and on the concentration of the protein. The careful control experiments must therefore be performed during investigations of proteins fused with EGFP, especially at pH lower than 7.
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Hu, Yu, Ziying Li, Wei Shi, Yanxue Yin, Heng Mei, Huafang Wang, Tao Guo, Jun Deng, Han Yan, and Xuan Lu. "Early diagnosis of cerebral thrombosis by EGFP–EGF1 protein conjugated ferroferric oxide magnetic nanoparticles." Journal of Biomaterials Applications 33, no. 9 (January 15, 2019): 1195–201. http://dx.doi.org/10.1177/0885328218823475.
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Cerebral thrombosis disease is a worldwide problem, with high rates of morbidity, disability, and mortality. Magnetic resonance imaging diffusion-weighted imaging was used as an important early diagnostic method for cerebral thrombotic diseases; however, its diagnosis time is 2 h after onset. In this study, we designed EGFP–EGF1–NP–Fe3O4 for earlier diagnosis of cerebral thrombosis by taking advantage of EGFP–EGF1 fusion protein, in which EGF1 can bind with tissue factor and enhanced green fluorescent protein has previously been widely used as a fluorescent protein marker. EGFP–EGF1–NP–Fe3O4 or NP–Fe3O4 reaches the highest concentration in the infarction areas in 1 h. To evaluate the targeting ability of EGFP–EGF1–NP–Fe3O4, a fluorochrome dye, Dir, was loaded into the nanoparticle. As shown by the in vivo organ multispectral fluorescence imaging, Dir-loaded EGFP–EGF1–NP–Fe3O4 exhibited higher fluorescence than those of model rats treated with Dir-loaded NP–Fe3O4. Coronal frozen sections and transmission electron microscope further showed that EGFP–EGF1–NP–Fe3O4 was mainly accumulated in the tissue factor exposure region of brain. The data indicated that the EGFP–EGF1–NP–Fe3O4 targeted cerebral thrombosis and might be applied in the early diagnosis of intracranial thrombosis.
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BOLSOVER, Stephen, Ozbek IBRAHIM, Niamh O'LUANAIGH, Helen WILLIAMS, and Shamshad COCKCROFT. "Use of fluorescent Ca2+ dyes with green fluorescent protein and its variants: problems and solutions." Biochemical Journal 356, no. 2 (May 24, 2001): 345–52. http://dx.doi.org/10.1042/bj3560345.
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We have studied the degree to which fluorescent Ca2+ indicator dyes, and green fluorescent protein and its variants, can be used together. We find that the most commonly used fluorescent protein, enhanced green fluorescent protein (EGFP), seriously contaminates fura 2 signals. We suggest two alternative combinations for which there is no detectable contamination of the Ca2+ indicator signal by the fluorescent protein. Blue fluorescent protein can be used with the Ca2+ indicator Fura Red; EGFP can be used with the Ca2+ indicator X-Rhod 1. The use of these combinations will permit the accurate measurement of Ca2+ signals in cells transfected with fluorescent proteins.
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Spitzer, Dirk, Kurt E. J. Dittmar, Manfred Rohde, Hansjörg Hauser, and Dagmar Wirth. "Green Fluorescent Protein-Tagged Retroviral Envelope Protein for Analysis of Virus-Cell Interactions." Journal of Virology 77, no. 10 (May 15, 2003): 6070–75. http://dx.doi.org/10.1128/jvi.77.10.6070-6075.2003.
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ABSTRACT Fluorescent retroviral envelope (Env) proteins were developed for direct visualization of viral particles. By fusing the enhanced green fluorescent protein (eGFP) to the N terminus of the amphotropic 4070A envelope protein, extracellular presentation of eGFP was achieved. Viruses incorporated the modified Env protein and efficiently infected cells. We used the GFP-tagged viruses for staining retrovirus receptor-positive cells, thereby circumventing indirect labeling techniques. By generating cells which conditionally expressed the GFP-tagged Env protein, we could confirm an inverse correlation between retroviral Env expression and infectivity (superinfection). eGFP-tagged virus particles are suitable for monitoring the dynamics of virus-cell interactions.
Dissertations / Theses on the topic "EGFP (enhanced green fluorescent protein)"
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Kumas, Gozde. "Detecting G-protein Coupled Receptor Interactions Using Enhanced Green Fluorescent Protein Reassembly." Master's thesis, METU, 2012. http://etd.lib.metu.edu.tr/upload/12614136/index.pdf.
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The largest class of cell surface receptors in mammalian genomes is the superfamily of G protein-coupled receptors (GPCRs) which are activated by a wide range of extracellular responses such as hormones, pheromones, odorants, and neurotransmitters. Drugs which have therapeutic effects on a wide range of diseases are act on GPCRs. In contrast to traditional idea, it is recently getting accepted that G-protein coupled receptors can form homo- and hetero-dimers and this interaction could have important role on maturation, internalization, function or/and pharmacology.
Bimolecular fluorescence complementation technique (BiFC)is an innovative approach based on the reassembly of protein fragments which directly report interactions. In our study we implemented this technique for detecting and visualizing the GPCR interactions in yeast cells. The enhanced green fluorescent protein (EGFP) fractionated into two fragments at genetic level which does not possess fluorescent function. The target proteins which are going to be tested in terms of interaction are modified with the non-functional fragments, to produce the fusion proteins. The interaction between two target proteins, in this study Ste2p receptors which are alpha pheromone receptors from Saccharomyces cerevisiae, enable the fragments to come in a close proximity and reassemble. After reassembly, EGFP regains its fluorescent function which provides a direct read-out for the detection of interaction. Further studies are required to determine subcellular localization of the interaction. Moreover, by using the fusion protein partners constructed in this study, effects of agonist/antagonist binding and post-translational modifications such as glycosylation and phosphorylation can be examined. Apart from all, optimized conditions for BiFC technique will guide for revealing new protein-protein interactions.
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Rane, Lukas. "Improving the temporal resolution of a microspectrometer for the study of the photophysics of enhanced green fluorescent protein." Thesis, KTH, Tillämpad fysik, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-300136.
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The use of fluorescent proteins as fluorescent markers has exploded over the last decades. In particular due to the development of advanced microscopy for live cell measurements, dynamic molecular studies down to single molecule levels and for superresolution microscopy. Many variants of fluorescent proteins exist with varying properties, such as emission color, photostability and brightness. These properties enable advanced applications, like timeresolved imaging or imaging below the diffraction limit. However, the photophysics of fluorescent proteins are complex and in many aspects quite unexplored. The triplet state in particular, is a central photophysical state because it is an entrance gate to an ensamble of deleterious photochemical processes that compromise the photostability of fluorescent proteins.The Pixel team at Institute de Biologie Structurale in France, is mainly focused on developing fluorescent proteins for advanced fluorescence imaging. One of the goals is to understand the influence of photochemistry on the properties of fluorescent proteins.In this project, a method to indirectly observe the triplet state in the prototypical EGFP fluorescent protein was developed. The introduction of new hardware and software, coupled to biophysical experiments, required an interdisciplinary strategy to tackle the obstacles during the route. Experiments under different environmental conditions to test the influence on the population of the triplet state of viscosity, pH, UV and infrared light, triplet state quenchers and temperature were performed.The results show that temperature and laser power greatly influence the triplet state kinetics in EGFP. Notably, it was found that the triplet state lifetime strongly increases at cryotemperature in comparison to roomtemperature. Overall, the newly developed setup and our preliminary results on EGFP open the door to novel studies on the photophysical properties of fluorescent proteins.Nyttjandet av fluorescerande proteiner som markörer har exploderat de senaste årtionden. Speciellt till följd av utvecklingen av avancerad mikroskopi för levande cellmätningar, dynamiska molekylära studier ned till enstaka molekylnivåer och för superupplösnings mikroskopi. Många varianter av fluorescerande proteiner förekommer med varierande egenskaper så som färg, fotostabilitet och ljusstyrka. Dessa proteiner möjliggör avancerade applikationer, som tidsupplöst bildgivning eller bildgivning med upplösning under diffraktionsgränsen. Fotofysiken bakom fluorescerande proteiner är komplex och i många aspekter ganska outforskad. Triplettillståndet är ett centralt fotofysiskt tillstånd eftersom det är en ingångsport till en rad skadliga fotokemiska processer som äventyrar fotostabiliteten hos fluorescerance proteiner.Pixelteamet på Institute de Biologie Structurale i Frankrike, fokuserar huvudsakligen på utveckling av fluorescerande proteiner för avancerad fluorescerande bildgivning. Ett av målen är att förstå hur fotokemi påverkar egenskaperna hos fluorescerande proteiner.I det här projektet har en metod för att indirekt observera triplettillståndet i det prototypiska fluorescerande proteinet EGFP utvecklats. Introduktionen av ny hårdvara och mjukvara, i kombination med biofysikaliska experiment, krävde en interdisiplinär strategi för att tackla utmaningarna under vägens gång. Experiment under olika miljömässiga förhållanden gjordes för att testa hur populationen av triplettillståndet påverkas till följd av viskositet, pH, UV och infrarött ljus, triplettillståndshämmare och temperatur.Resultaten visar att temperatur och lasereffekt har en stor påverkan på triplettillståndet och dess kinetik hos EGFP. Noterbart är att triplettillståndets livstid ökar kraftigt i kryotemperatur i jämförelse med rumstemperatur. Sammanfattningsvis så utvecklades en ny experimentel uppställning och de tidiga resultaten från EGFP har öppnat dörren för nya studier rörande de fotofysiska egenskaperna hos fluorescerande proteiner.
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Sousa, Ana Paula Abuchain. "Aumento de escala para a produção de Proteína verde fluorescente melhorada (Enhanced Green Fluorescent Protein - EGFP) a partir de Escherichia coli recombinante em biorreator convencional /." Araraquara, 2019. http://hdl.handle.net/11449/181957.
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Orientador: Marcel Otavio CerriResumo: Avanços na biotecnologia proporcionaram possibilidades para o eficiente desempenho da produção em larga escala de diversas biomoléculas e consequentemente suas aplicações industriais. A Escherichia coli se destaca dentre a gama de microrganismos que agem como hospedeiros de genes, desempenhando a função de codificar a síntese proteica. Os vetores mais veiculados na produção de proteínas recombinantes em E. coli são baseados no operon lac, onde o isopropil β-D1-tio-galactopiranosídeo (IPTG), análogo a molécula de lactose, é utilizado para a indução da produção da proteína de interesse. Estudos descritos na literatura também observaram o bom desempenho da lactose como agente de indução da E. coli recombinante na expressão de proteína verde fluorescente melhorada (Enhanced Green Fluorescent Protein – EGFP), e suas vantagens quando comparada ao IPTG, como por exemplo menor custo e menor toxicidade. A EGFP se tornou promissora pelo fato de ser monomérica e não precisar de auxilio de quaisquer agentes adicionais para exibir atividade de fluorescência. Possui variadas utilidades no campo biológico como excelente biomarcador da expressão genica e biosensor. Em bioprocessos, operados em biorreatores convencionais é fundamental o estudo dos parâmetros interferentes nos cultivos para a otimização da expressão do produto desejado. A oxigenação em processos conduzidos de maneira aeróbica, também é uma tarefa desafiadora em biorreatores convencionais, sendo imprescindível o controle da con... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Advances in biotechnology have provided possibilities to the performance of large-scale of biomolecules and therefore of their industrial applications. Escherichia coli stands out among microorganisms that act as host genes, functioning as a synthetic protein. The most useful vectors for the production of recombinant proteins in E. coli are based on the operon lac, where isopropyl β-D1-thiogalactopyroside (IPTG), analogous to lactose, is used to induce the production of proteins of interest. Studies in the literature have also observed the good performance of lactose as an inducer of recombinant E. coli in the expression of Enhanced Green Fluorescent Protein (EGFP), and its advantages when compared to IPTG, such as lower cost and toxicity. EGFP becomes promising because it is monomeric and does not need to help the main agents for fluorescence activity. It has several uses in the biological field as an excellent biomarker of gene expression and biosensor. In bioprocesses, operated in conventional bioreactors, it is fundamental to study the interfering parameters in the cultures to optimize the expression of the desired product. Oxygenation in aerobically conducted processes is also a challenging task in conventional bioreactors, with control of the concentration of dissolved oxygen in the culture medium is essential, which may be limiting for growth and expression of the protein of interest. In processes of production of heterologous proteins, it is assumed that the productiv... (Complete abstract click electronic access below)
Mestre
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Kirberger, Michael Patrick. "Analyses and Applications of Metalloprotein Complexes." Digital Archive @ GSU, 2008. http://digitalarchive.gsu.edu/chemistry_theses/14.
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The structural characteristics associated with the binding of beneficial metals (i.e. - Mg2+, Zn2+ and Ca2+) to natural proteins has typically received more attention than competitive binding by toxic metals (e.g. – Pb2+, Hg2+, Cd2+, La3+, etc.). In this thesis, a statistical analysis of Pb2+-binding in crystallized protein structures indicates that Pb2+ does not bind preferentially with nitrogen, as generally assumed, but binds predominantly with oxygen, and to a lesser degree, sulfur. A comparison of Ca2+ and Pb2+ indicates that Pb2+ binds with a wider range of coordination numbers, with less formal change, and with less defined structure than Ca2+. The Pb2+ ion also appears to displace Ca2+ with little conformational stress in calcium binding proteins (CaBP’s). Experimental data from the binding of metals with engineered fluorescent proteins indicate that both Pb2+ and Gd3+ will occupy grafted calcium-binding sites with greater affinity than Ca2+, and strong evidence is presented to support the hypothesis that Pb2+ and Gd3+ will bind non-specifically on the protein surface. These results suggest that toxicity is associated with two binding mechanisms: displacement of the metal cofactor which disrupts protein function, and non-specific binding which maintains higher solubility of the metal.
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Chen, X. "TAGGING BIOCONTROL STREPTOMYCES TO STUDY LETTUCE COLONIZATION." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/345187.
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The ability of the biological control agents (BCAs) to colonize plant tissues is an important feature involved in microbe-assisted plant protection. Plant-microbe interaction research increased especially in the last decade thanks to technological revolution. Molecular methods and the development of advanced microscopic techniques allow researchers to explore gene expression and localization of beneficial microorganisms within plants. The green fluorescent protein (GFP) and its modified version, enhanced GFP (EGFP), more adapt for expression in mammalian cells and GC-rich actinomycetes like Streptomyces, have been widely used as markers to study gene expression, as well as plant-microbe interactions. Aside fluorescent protein approaches, fluorescence in situ hybridization (FISH) is another frequently used technique to visualize microbial colonization patterns and community composition by application of specific fluorescent probes. Firstly, we transformed five Streptomyces strains, which showed strong inhibition activity against Sclerotinia sclerotiorum, with the EGFP construct by the conjugation method. The conjugation efficiencies varied between the strains, but were comparable to the reference strain. The fitness of transformed strains was similar to wild-type; the transformants maintained similar sporulation, mycelium growth rate, and the ability to produce important secondary metabolites and lytic enzymes. Secondly, two transformed strains, Streptomyces cyaneus ZEA17I, and Streptomyces sp. SW06W, were used to study lettuce colonization dynamics by seed coating method. Their spatio-temporal dynamics were determined in sterile substrate. The strains were consistently recovered from lettuce rhizosphere and inner root tissues up to six weeks. Finally, the colonization pattern of lettuce by Streptomyces cyaneus ZEA17I was examined by both EGFP and FISH approaches combined with confocal laser scanning microscopy (CLSM). For FISH-CLSM analysis, universal bacteria and Streptomyces genus specific probes were used to label S. cyaneus ZEA17I. The consistent presence of the labeled strain at the lettuce root one week after sowing showed that Streptomyces spores could rapidly germinate and produce filamentous mycelium on lettuce. S. cyaneus ZEA17I was detected also on two-week-old roots, indicating the long-term survival ability of this strain in lettuce rhizosphere. Altogether, the antagonistic activity, rhizosphere and root competence showed by the Streptomyces conferred their potential to act as BCA. Further studies on the complex host-pathogen-antagonist interactions will provide additional knowledge to understand the modes and mechanisms of Streptomyces-mediated plant protection.
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McRae, Shelley Rose. "Green Fluorescent Proteins: Towards Extra-Cellular Applications?" Thesis, Griffith University, 2009. http://hdl.handle.net/10072/368106.
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The Green Fluorescent Protein (GFP) and its numerous variants are applied
extensively in a multitude of in vivo applications and have been studied in this context
at length. In contrast however, the study of GFP’s within the emerging fields of nanoand
micro-technology, which offer broader extracellular applications for GFP and its
derivatives, has only recently begun to gather momentum. This thesis presents the
directed design of a novel series of Enhanced Green and Enhanced Yellow Fluorescent
Proteins (EGFP and EYFP respectively), for implementation in extracellular
applications such as biosensing and fundamental research into fluorescence protein
behaviour. Each parent fluorescent protein (EGFP or EYFP) was altered to display a
single solvent exposed reactive sulfhydryl group with varying degrees of connectivity to
the internal GFP chromophore. These sulfhydryl groups were introduced into the
protein primary structure via point mutation to yield cysteine residues in place of the
targeted native amino acid. Careful examination of the EGFP and EYFP tertiary
structures to identify amino acids within the protein primary sequence that fulfilled
specific criteria, which were defined in our experimental design, resulted in substitution
of amino acids at positions 221, 223, 219, 212 and 97 in EGFP and 221, 223, 212, 95
and 21 in EYFP.
Critical development of supporting methodologies delivered vast improvements
on literature protocols for expression and purification of the GFP variants listed above.
Expression protocol investigation determined that the most prolific E. coli strain for
recombinant fluorescent protein production was BL21, which, coupled with our
methodology, produced up to 13.6 mg of fluorescent protein per gram of wet cell pellet.
The novel purification procedure described in this Thesis delivered highly pure protein
with impressive yields (75-80 %).
Characterisation of the novel proteins that were designed and produced during
this work revealed no change in the proteins’ ability to resist denaturation resulting from
cysteine substitution. Neither was there any change in fluorescence emission or UV
absorption profiles for standard concentrations (< 60 mM) of any of the purified proteins
that were produced. While standard protein solutions returned normal fluorescence
emission profiles, solutions that contained protein concentrations above 60 mM
displayed red shifted emission maximum values. For protein solutions within the mM
concentration range this red shift in fluorescence emission was at times in the order of
30 nm resulting in emission maximums of up to 540 nm for EGFP, and 548 nm for
EYFP and recombinant proteins containing an L221C mutation. Preliminary
investigations into this phenomenon showed that the changes observed in fluorescence
emission were dependent on protein concentration and could be due to dipole-dipole
interactions which may be induced by protein aggregate formation at high protein
concentrations. Manipulations that were performed on fluorescent proteins during this
study included proteolytic digestion with Proteinase K and subsequent testing of the
digested protein product. This work identified an increase in the quantum yield of
proteolytically digested EGFP and EYFP from 0.6 to 0.8 accompanied by no reduction
in the digested proteins resistance to denaturing treatments except when treated with
1 % SDS solution.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
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Birk, Stephanie. "Genetische Markierung von humanen mesenchymalen Stammzellen mittels enhanced green fluorescent protein." Diss., kostenfrei, 2008. http://edoc.ub.uni-muenchen.de/8123/.
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Fan, Zhenchuan Bird R. Curtis. "Development of a recombinant noncytopathic bovine viral diarrhea virus stably expressing enhanced green fluorescent protein." Auburn, Ala., 2005. http://repo.lib.auburn.edu/2005%20Summer/master's/FAN_ZHENCHUAN_4.pdf.
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Walker, Wendilywn E. "Towards gene therapy for cystic fibrosis : enhanced green fluorescent protein as a reporter of promoter activity." Thesis, University of Edinburgh, 2005. http://hdl.handle.net/1842/27597.
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The aim of this project was to investigate the use of the Enhanced Green Fluorescent Protein (EGFP) as a reporter of CFTR promoter activity. six vectors were created coupling portions of the CFTR locus to EGFP in GCVs. Small plasmids were made by conventional cloning procedures, while large PAC vectors were made by a double recombination method employing both homologous and Cre recombinase/loxP recombination. These vectors were transfected into permanent cell lines COS7, MDCK-iowa, T84 and CaCO2, in order to assess the effects of the genomic context elements upon EGFP. The proximal CFTR 5’ region in the p1kbcfproEGFP vector drove expression of the EGFP transgene at low levels in every cell line analysed. This is in agreement with previous reports that show basal levels of CFTR expression driven by this proximal ‘housekeeping’ region. The additional upstream region in the PAC65bcfproEGFP vector did not appear to modulate expression in any of the cell lines analysed. A comparison of the twin vectors PACRC1iresEGFP and PACRC2iresEGFP, which differ only in the absence or presence of CFTR intron 1 respectively, showed similar levels of expression in the COS7 and MDCK-iowa cell lines. Thus, the intron 1 element does not seem to alter expression in these non-gut cell lines; this is consistent with reports that show regulation of CFTR expression in response to the intron element to be specific to cells of the gut epithelium. A comparison of pEGFP-N and PACRC2cmvEGFP revealed that large PAC vectors show an intrinsic reduction in expression in comparison to their small plasmid counterparts. Further experiments showed that this was not an effect of vector copy number, and that the effect could not act in trans upon a co-transfected molecule. These studies also revealed an unexpected interaction: diluting a reporter plasmid with an anonymous plasmid may actually increase its transfection efficiency. An ex vivo primary air interface sheep tracheal culture was utilised as a more realistic model. Cultures were transfected with several of the genomic context vectors. While PAC vectors had shown a dramatic reduction in expression relative to their small plasmid counterparts in the in vitro studies, only a small reduction was seen to the ex vivo cultures, thus PAC vectors, such as GCVs, may provide a promising approach for gene therapy studies.
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Masters, T. A. "Time-resolved fluorescence studies of enhanced green fluorescent protein and the molecular dynamics of 3-Phosphoinositide Dependent Protein Kinase 1." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/19031/.
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Fluorescent proteins (FPs), particularly Enhanced Green Fluorescent Protein (EGFP), are essential tools in the study of intact biological systems. Whilst the photophysics of its progenitor, GFP, have been investigated extensively, far fewer studies of EGFP have been made. In this thesis, a full characterisation of EGFP excited state photophysics by singleand two-photon time-resolved fluorescence lifetime and anisotropy is presented. Furthermore, the two-photon transition tensor, determined by absorption and initial anisotropies, is shown to be dominated by a single element. The two-photon excited state of EGFP was subject to Stimulated Emission Depletion (STED), revealing the stimulated emission cross section, the ground state relaxation time and the time evolution of the higher order distribution moments to which anisotropy is not sensitive. The strong adherence to theoretical Debye diffusion reinforced the conclusions of the two-photon structure model, and showed EGFP to be an excellent molecule for the future development of STED. In addition, these studies provided a sound basis on which to employ single- and two-photon FRET in vivo and in vitro. Cell behaviour is governed by the transduction of molecular signals from the extracellular environment to intracellular compartments. At the centre of the PI 3-kinase signalling pathway is PDK1, a Serine/Threonine kinase, which phosphorylates numerous important downstream targets including Protein Kinase B (PKB). To date however, the regulatory mechanisms governing the behaviour of this protein remain poorly understood. Timeresolved fluorescence lifetime imaging microscopy (FLIM) was employed with FP tagged PDK1 to investigate dynamic interactions in intact cells in situ and in vivo. PDK1 was shown to dimerise in a manner dependent on PI 3-kinase activity and PDK1 PH domain lipid binding. To detail the structure of the observed intermolecular interaction, recombinant FP labelled PDK1 was produced with insect cells. Measurement of the rise in acceptor fluorescence during FRET in vitro indicated the PDK1 dimer pair exists in an antiparallel arrangement. These results provide the first insight on the structure of the dimer and demonstrate that the generation of 3-phosphorylated lipids is required for its formation.
Book chapters on the topic "EGFP (enhanced green fluorescent protein)"
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Baumstark-Khan, Ch, Ch E. Hellweg, and G. Horneck. "On the Suitability of Red and Enhanced Green Fluorescent Protein (DsRed/EGFP) as Reporter Combination." In Coupling of Biological and Electronic Systems, 1–13. Berlin, Heidelberg: Springer Berlin Heidelberg, 2002. http://dx.doi.org/10.1007/978-3-642-56177-1_1.
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Utratna, Marta, and Conor P. O’Byrne. "Using Enhanced Green Fluorescent Protein (EGFP) Promoter Fusions to Study Gene Regulation at Single Cell and Population Levels." In Methods in Molecular Biology, 233–47. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-0703-8_20.
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"eGFP (enhanced green fluorescent protein)." In Encyclopedia of Genetics, Genomics, Proteomics and Informatics, 585. Dordrecht: Springer Netherlands, 2008. http://dx.doi.org/10.1007/978-1-4020-6754-9_5121.
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Zhao, Xiaoning, Xin Jiang, Chiao-Chian Huang, Steven R. Kain, and Xianqiang Li. "[36] Generation of a destabilized form of enhanced green fluorescent protein." In Methods in Enzymology, 438–44. Elsevier, 1999. http://dx.doi.org/10.1016/s0076-6879(99)02038-8.
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Kain, Steven R., and Jing-Tyan Ma. "[5] Early detection of apoptosis with annexin V-enhanced green fluorescent protein." In Methods in Enzymology, 38–43. Elsevier, 1999. http://dx.doi.org/10.1016/s0076-6879(99)02007-8.
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KAIN, STEVEN R. "Enhanced Variants of the Green Fluorescent Protein for Greater Sensitivity, Different Colours and Detection of Apoptosis." In Fluorescent and Luminescent Probes for Biological Activity, 284–92. Elsevier, 1999. http://dx.doi.org/10.1016/b978-012447836-7/50021-x.
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Zhao, Xiaoning, Tommy Duong, Chiao-Chian Huang, Steven R. Kain, and Xianoiang Li. "[4] Comparison of enhanced green fluorescent protein and its destabilized form as transcription reporters." In Methods in Enzymology, 32–38. Elsevier, 1999. http://dx.doi.org/10.1016/s0076-6879(99)02006-6.
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Fang, Yu, Chiao-chian Huang, Steven R. Kain, and Xianqiang Li. "[18] Use of coexpressed enhanced green fluorescent protein as a marker for identifying transfected cells." In Methods in Enzymology, 207–12. Elsevier, 1999. http://dx.doi.org/10.1016/s0076-6879(99)02020-0.
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Ishii, T., M. Hayashi, M. Takagi, A. Suwanai, S. Narumi, S. Suzuki, Y. Matsuzaki, KL Parker, and T. Hasegawa. "A Genome-Wide Expression Profile of Steroidogenic Cells Selectively Derived from Adrenal Glands of Knockout Mice Lacking Steroidogenic Acute Regulatory Protein by Targeted Expression of Enhanced Green Fluorescent Protein." In Posters I, P3–20—P3–20. Endocrine Society, 2010. http://dx.doi.org/10.1210/endo-meetings.2010.part3.p1.p3-20.
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Vergara, Ricardo, Felipe Olivares, Blanca Olmedo, Carolina Toro, Marisol Muñoz, Carolina Zúñiga, Roxana Mora, et al. "Gene Editing in Prunus Spp.: The Challenge of Adapting Regular Gene Transfer Procedures for Precision Breeding." In Prunus - Recent Advances [Working Title]. IntechOpen, 2021. http://dx.doi.org/10.5772/intechopen.98843.
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Successfully gene editing (GE) in Prunus spp. has been delayed due to its woody nature presenting additional difficulties in both, proper regeneration protocols and designing efficient gene transfer techniques. The availability of adequate, single cell culture techniques for GE such as protoplast regeneration, is a limiting step for the genus and for this reason, the improvement of regular regeneration protocols and finding more efficient techniques for the delivery of the “editing reagents” seem to be a reasonable strategy to incorporate GE in the genus. During the last 10 years, we have focused our efforts optimizing some previous regeneration and gene transfer procedures for Japanese plum (P. salicina), sweet cherry (P. avium) and peach (P. persica) to incorporate them into a GE technology on these species. In parallel, delivery techniques for the CRISPR/Cas9 editing components, i.e., guide RNA (gRNA) and Cas9, have been developed with the aim of improving gene targeting efficiencies. In that line, using DNA virus-based replicons provides a significant improvement, as their replicational release from their carriers enables their enhanced expression. Here, we make a brief overview of the tissue culture and regeneration protocols we have developed for P. salicina, P. avium and P. persica, and then we proceed to describe the use of Bean yellow dwarf virus (BeYDV)-derived replicon vectors to express the editing reagents in vivo and to evaluate their editing capability on individuals derived from Agrobacterium-mediated gene transfer experiments of these species. We show part of our characterization assays using new BeYDV-derived vectors harboring multiple gRNAs, the Cas9 gene, and the green fluorescent protein reporter gene. We also describe a dedicated genome analysis tool, by which gRNA pairs can be designed to address gene deletions of the target genes and to predict off-target sequences. Finally, as an example, we show the general results describing GE of the peach TERMINAL FLOWER 1 gene and some preliminary characterizations of these materials.
Conference papers on the topic "EGFP (enhanced green fluorescent protein)"
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Hofkens, Johan, Frans C. De Schryver, Mircea Cotlet, and Satoshi Habuchi. "Single molecule surface enhanced resonance Raman scattering (SERRS) of the enhanced green fluorescent protein (EGFP)." In Biomedical Optics 2004, edited by Alexander P. Savitsky, Lubov Y. Brovko, Darryl J. Bornhop, Ramesh Raghavachari, and Samuel I. Achilefu. SPIE, 2004. http://dx.doi.org/10.1117/12.531307.
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Chou, Yan-Syun, and Yu-kaung Chang. "Stirred Fluidized Bed Immobilized Metal Affinity Chromatography for Direct Recovery of Poly His-tagged Enhanced Green Fluorescent Protein." In 14th Asia Pacific Confederation of Chemical Engineering Congress. Singapore: Research Publishing Services, 2012. http://dx.doi.org/10.3850/978-981-07-1445-1_709.
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Tchebotarev, L., and L. Valentovich. "Engineering of vectors essential to derive chimeric proteins based on superfolder green fluorescent protein and harpins." In 2nd International Scientific Conference "Plants and Microbes: the Future of Biotechnology". PLAMIC2020 Organizing committee, 2020. http://dx.doi.org/10.28983/plamic2020.245.
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Conjugation of harpins with green fluorescent protein is aimed at achieving enhanced solubility and stability of chimeric protein, facilitating qualitative and quantitative evaluation of its expression in the routine experiments.
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Ren, Z. F. "Nano Materials and Physics." In ASME 4th Integrated Nanosystems Conference. ASMEDC, 2005. http://dx.doi.org/10.1115/nano2005-87045.
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Aligning carbon nanotubes in any way desired is very important for many fundamental and applied research projects. In this talk, I will first discuss how to grow them with controlled diameter, length, spacing, and periodicity using catalyst prepared by magnetron sputtering, electron beam (e-beam) lithography, electrochemical deposition, and nanosphere self-assembly. Then I will present our results of field emission property of both the aligned carbon nanotubes grown on flat substrates and random carbon nanotubes grown on carbon cloth. For the aligned carbon nanotubes arrays, I will present the preliminary results of using them as photonic band gap crystals and nanoantennae. As an alternative material of carbon nanotubes, ZnO nanowires have been grown in both aligned fashion on flat substrates and random fashion on carbon cloth. Using these ZnO nanowires, good field emission properties were observed. Furthermore, I will present our recent studies on the electrical breakdown and transport properties of a single suspended nanotube grown on carbon cloth by a scanning electron microscope probe incorporated into a high resolution transmission electron microscope. As part of the potential applications, I will also discuss our recent success on molecules delivery into cells using carbon nanotubes. Finally I will talk about our most recent endeavor on achieving thermoelectric figure-of-merit (ZT) higher than 2 using our unique nanocomposite approach. Plasma-enhanced chemical vapor deposition (PECVD) was discovered by my group in 1998 to be able to grow aligned carbon nanotubes [1]. Catalyst film was first deposited by magnetron sputtering. According to the thickness of the catalytic film, aligned carbon nanotubes were grown with different diameters and spacing, and different length depending the growth time. However, the two major drawbacks are 1) that the location of where the nantoube grows can not be controlled, 2) that the spacing between the nanotubes can not be varied too much. Therefore, we immediately explored to grow aligned carbon nanotubes with the location and spacing controls using e-beam lithography [2]. Unfortunately the cost is so high that the e-beam is not suited for large scale commercialization that requires only an average site density control not the exactly location, for example, electron source. It is the cost issue that made us to develop electrochemical deposition to make catalyst dots that can be separated more than 10 micormeters between dots [3]. With such arrays, we tested many samples for field emission properties and found the optimal site density [4]. However, for applications that require the location controls, for example, photonic band gap crystals, electrochemical deposition can not be satisfactory. It is this kind of application that led us to develop the nanosphere self-assembly technique in large scale [5]. For field emission, we found that ZnO nanowires are good field emitters comparable to carbon nanotubes if they are grown with the right diameter and spacing. Here I will discuss the field emission properties of ZnO nanowires as an alternative material to carbon nanotubes [6]. Us a special kind of carbon nanotubes made by PECVD, we discovered a highly efficient molecular delivery technique, named nanotube spearing, based on the penetration of Ni-particle embedded nanotubes into cell membranes by magnetic field driving. DNA plasmids encoding the enhanced green fluorescent protein (EGFP) sequence were immobilized onto the nanotubes, and subsequently speared into targeted cells. We have achieved the unprecedented high transduction efficiency in Bal17 B-lymphoma, ex vivo B cells, and primary neurons with high viability. This technique may provide a powerful tool for high efficient gene transfer in a variety of cells, especially, the hard-to-transfect cells [7]. Conventional transport studies of multiwall carbon nanotubes (MWNTs) with only the outmost wall contacted to the electrodes via side-contact shows that a MWNT is a ballistic conductor with only the outmost wall carrying current. Here we show, by using end-contact in which every wall is contacted to the electrodes, that every wall is conducting, as evidenced by a significant amount of current drop when an innermost wall is broken at high-bias. Remarkably, the breakdown of each wall was initiated in the middle of the nanotube, not at the contacts, indicating diffusive electron transport. Using end-contact, we were able to probe the conductivity wall-by-wall and found that each wall is indeed either metallic, or semiconducting, or pseudogap-like. These findings not only reveal the intrinsic physical properties of MWNTs but also provide important guidance to MWNT-based electronic devices [8]. At the end of the talk, if time permits, I will talk about our ongoing effort on improving the figure-of-merit (ZT) of thermoelectric materials using a nanocomposite strategy to mimic the structure of the superlattice of PbTe/PbSe and Bi2Te3/Sb2Te3 hoping to reduce the thermal conductivity by a factor of 2–4 while maintaining the electrical conductivity. To make a close to 100% dense nanocomposite, we started with nanoparticles synthesis, then consolidation using both the traditional hot press and the direct current fast-heat, named plasma pressure compact, to preserve the nano size of the component particles. So far, we have seen thermal conductivity decrease by a factor of 2 in the systems of Si/Ge, PbeTe/PbSe, Bi2Te3/Sb2Te3, indicating the potential of improving ZT by a factor of 2.
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Cotlet, Mircea, Peter M. Goodwin, Geoffrey S. Waldo, and James H. Werner. "Time-resolved detection of the one- and two-photon excited fluorescence of single molecules of a folding enhanced green fluorescent protein." In Biomedical Optics 2006, edited by Jörg Enderlein and Zygmunt K. Gryczynski. SPIE, 2006. http://dx.doi.org/10.1117/12.646697.
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