6,875 research outputs found

    Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

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    Background: Homologous recombination mediated by the lambda-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the lambda-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these lambda-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains. \ud \ud Results: Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the lambda-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6xHis, 3xFLAG, 4xProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the lambda-Red system, which can lead to unwanted secondary alterations to the chromosome. \ud \ud Conclusion: We have developed a counter-selective recombineering technique for epitope tagging or for deleting genes in E. coli. We have demonstrated the versatility of the technique by modifying the chromosome of the enterohaemorrhagic O157:H7 (EHEC), uropathogenic CFT073 (UPEC), enteroaggregative O42 (EAEC) and enterotoxigenic H10407 (ETEC) E. coli strains as well as in K-12 laboratory strains

    Dynamic criticality in glass-forming liquids

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    We propose that the dynamics of supercooled liquids and the formation of glasses can be understood from the existence of a zero temperature dynamical critical point. To support our proposal, we derive from simple physical assumptions a dynamic field theory for supercooled liquids, which we study using the renormalization group (RG). Its long time behaviour is dominated by a zero temperature critical point, which for dimensions d > 2 belongs to the directed percolation universality class. Molecular dynamics simulations confirm the existence of dynamic scaling behaviour consistent with the RG predictions.Comment: 4 pages, 2 figure

    Culex tarsalis is a competent vector species for Cache Valley virus

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    Background: Cache Valley virus (CVV) is a mosquito-borne orthobunyavirus endemic in North America. The virus is an important agricultural pathogen leading to abortion and embryonic lethality in ruminant species, especially sheep. The importance of CVV in human public health has recently increased because of the report of severe neurotropic diseases. However, mosquito species responsible for transmission of the virus to humans remain to be determined. In this study, vector competence of three Culex species mosquitoes of public health importance, Culex pipiens, Cx. tarsalis and Cx. quinquefasciatus, was determined in order to identify potential bridge vector species responsible for the transmission of CVV from viremic vertebrate hosts to humans. Results: Variation of susceptibility to CVV was observed among selected Culex species mosquitoes tested in this study. Per os infection resulted in the establishment of infection and dissemination in Culex tarsalis, whereas Cx. pipiens and Cx. quinquefasciatus were highly refractory to CVV. Detection of viral RNA in saliva collected from infected Cx. tarsalis provided evidence supporting its role as a competent vector. Conclusions: Our study provided further understanding of the transmission cycles of CVV and identifies Cx. tarsalis as a competent vector

    Dephasing Times in a Non-degenerate Two-Dimensional Electron Gas

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    Studies of weak localization by scattering from vapor atoms for electrons on a liquid helium surface are reported. There are three contributions to the dephasing time. Dephasing by the motion of vapor atoms perpendicular to the surface is studied by varying the holding field to change the characteristic width of the electron layer at the surface. A change in vapor density alters the quasi-elastic scattering length and the dephasing due to the motion of atoms both perpendicular and parallel to the surface. Dephasing due to the electron-electron interaction is dependent on the electron density.Comment: 4 pages, Revte

    Gravitational Waves from Core Collapse Supernovae

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    We present the gravitational wave signatures for a suite of axisymmetric core collapse supernova models with progenitors masses between 12 and 25 solar masses. These models are distinguished by the fact they explode and contain essential physics (in particular, multi-frequency neutrino transport and general relativity) needed for a more realistic description. Thus, we are able to compute complete waveforms (i.e., through explosion) based on non-parameterized, first-principles models. This is essential if the waveform amplitudes and time scales are to be computed more precisely. Fourier decomposition shows that the gravitational wave signals we predict should be observable by AdvLIGO across the range of progenitors considered here. The fundamental limitation of these models is in their imposition of axisymmetry. Further progress will require counterpart three-dimensional models.Comment: 10 pages, 5 figure

    Electrical properties of Bi-implanted amorphous chalcogenide films

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    The impact of Bi implantation on the conductivity and the thermopower of amorphous chalcogenide films is investigated. Incorporation of Bi in Ge-Sb-Te and GeTe results in enhanced conductivity. The negative Seebeck coefficient confirms onset of the electron conductivity in GeTe implanted with Bi at a dose of 2x1016 cm-2. The enhanced conductivity is accompanied by defect accumulation in the films upon implantation as is inferred by using analysis of the space-charge limited current. The results indicate that native coordination defects in lone-pair semiconductors can be deactivated by means of ion implantation, and higher conductivity of the films stems from additional electrically active defects created by implantation of bismuth.Comment: This is an extended version of the results presented in Proc. SPIE 8982, 898213 (2014

    Miniature Toroidal Radio Frequency Ion Trap Mass Analyzer

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    A miniature ion trap mass analyzer is reported. The described analyzer is a 1/5-scale version of a previously reported toroidal radio frequency (rf) ion trap mass analyzer. The toroidal ion trap operates with maximum rf trapping voltages about 1 kVp-p or less; however despite the reduced dimensions, it retains roughly the same ion trapping capacity as conventional 3D quadrupole ion traps. The curved geometry provides for a compact mass analyzer. Unit-mass resolved mass spectra for n-butylbenzene, xenon, and naphthalene are reported and preliminary sensitivity data are shown for naphthalene. The expected linear mass scale with rf amplitude scan is obtained when scanned using a conventional mass-selective instability scan mode combined with resonance ejection

    Wave Propagation in Stochastic Spacetimes: Localization, Amplification and Particle Creation

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    Here we study novel effects associated with electromagnetic wave propagation in a Robertson-Walker universe and the Schwarzschild spacetime with a small amount of metric stochasticity. We find that localization of electromagnetic waves occurs in a Robertson-Walker universe with time-independent metric stochasticity, while time-dependent metric stochasticity induces exponential instability in the particle production rate. For the Schwarzschild metric, time-independent randomness can decrease the total luminosity of Hawking radiation due to multiple scattering of waves outside the black hole and gives rise to event horizon fluctuations and thus fluctuations in the Hawking temperature.Comment: 26 pages, 1 Postscript figure, submitted to Phys. Rev. D on July 29, 199
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