Producing gene deletions in Escherichia coli by P1 transduction with excisable antibiotic resistance cassettes

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion

PT Symmetric, Hermitian and P-Self-Adjoint Operators Related to Potentials in PT Quantum Mechanics

In the recent years a generalization $H=p^2 +x^2(ix)^\epsilon$ of the harmonic oscillator using a complex deformation was investigated, where \epsilon\ is a real parameter. Here, we will consider the most simple case: \epsilon even and x real. We will give a complete characterization of three different classes of operators associated with the differential expression H: The class of all self-adjoint (Hermitian) operators, the class of all PT symmetric operators and the class of all P-self-adjoint operators. Surprisingly, some of the PT symmetric operators associated to this expression have no resolvent set

Bacterial autoaggregation

Many bacteria, both environmental and pathogenic, exhibit the property of autoaggregation. In autoaggregation (sometimes also called autoagglutination or flocculation), bacteria of the same type form multicellular clumps that eventually settle at the bottom of culture tubes. Autoaggregation is generally mediated by self-recognising surface structures, such as proteins and exopolysaccharides, which we term collectively as autoagglutinins. Although a widespread phenomenon, in most cases the function of autoaggregation is poorly understood, though there is evidence to show that aggregating bacteria are protected from environmental stresses or host responses. Autoaggregation is also often among the first steps in forming biofilms. Here, we review the current knowledge on autoaggregation, the role of autoaggregation in biofilm formation and pathogenesis, and molecular mechanisms leading to aggregation using specific examples

Development and test results of a readout chip for the GERDA experiment

This paper describes the F-CSA104 architecture and its measurement results. The F-CSA104 is for γ spectroscopy with Ge detectors. It is a low noise, fully integrated, four channel XFAB 0.6μm CMOS technology ASIC, that has been developed for the GERDA experiment. Each channel contains a charge sensitive preamplifier (CSA) followed by a 11.7MHz differential line driver. It has been particularly designed to operate in liquid argon (T = 87K/-186°C) and to have a measuring sensitivity of 660e- with an ENC of 110e-, after offline filtering with 10μs shaping, when connected to a 30pF load. Special techniques are used to improve the SNR such as a large input PMOS FET, an integrated 500MΩ CSA feedback resistor and a noise degeneration drain resistor

LUX -- A Laser-Plasma Driven Undulator Beamline

The LUX beamline is a novel type of laser-plasma accelerator. Building on the joint expertise of the University of Hamburg and DESY the beamline was carefully designed to combine state-of-the-art expertise in laser-plasma acceleration with the latest advances in accelerator technology and beam diagnostics. LUX introduces a paradigm change moving from single-shot demonstration experiments towards available, stable and controllable accelerator operation. Here, we discuss the general design concepts of LUX and present first critical milestones that have recently been achieved, including the generation of electron beams at the repetition rate of up to 5 Hz with energies above 600 MeV and the generation of spontaneous undulator radiation at a wavelength well below 9 nm.Comment: submitte

Testing the Master Constraint Programme for Loop Quantum Gravity III. SL(2,R) Models

This is the third paper in our series of five in which we test the Master Constraint Programme for solving the Hamiltonian constraint in Loop Quantum Gravity. In this work we analyze models which, despite the fact that the phase space is finite dimensional, are much more complicated than in the second paper: These are systems with an SL(2,\Rl) gauge symmetry and the complications arise because non -- compact semisimple Lie groups are not amenable (have no finite translation invariant measure). This leads to severe obstacles in the refined algebraic quantization programme (group averaging) and we see a trace of that in the fact that the spectrum of the Master Constraint does not contain the point zero. However, the minimum of the spectrum is of order $\hbar^2$ which can be interpreted as a normal ordering constant arising from first class constraints (while second class systems lead to $\hbar$ normal ordering constants). The physical Hilbert space can then be be obtained after subtracting this normal ordering correction.Comment: 33 pages, no figure

Enhanced Radiation Hardness and Faster Front Ends for the Beetle Readout Chip

This paper summarizes the recent progress in the development of the 128 channel pipelined readout chip Beetle, which is intended for the silicon vertex detector, the inner tracker, the pile-up veto trigger and the RICH detectors of LHCb. Deficiencies found in the front end of the Beetle Version 1.0 and 1.1 chips resulted in the submissions of BeetleFE 1.1 and BeetleFE 1.2, while BeetleSR 1.0 implements test circuits to provide future Beetle chips with logic circuits hardened against single event upset (SEU). Section I. motivates the development of new front ends for the Beetle chip, and section II. summarizes their concepts and construction. Section III. reports preliminary results from the BeetleFE 1.1 and BeetleFE 1.2 chips, while section IV. describes the BeetleSR 1.0 chip. An outlook on future test and development of the Beetle chip is given in section V

Comparison of type 5d autotransporter phospholipases demonstrates a correlation between high activity and intracellular pathogenic lifestyle

Autotransporters, or type 5 secretion systems, are widespread surface proteins of Gram-negative bacteria often associated with virulence functions. Autotransporters consist of an outer membrane β-barrel domain and an exported passenger. In the poorly studied type 5d subclass, the passenger is a patatin-like lipase. The prototype of this secretion pathway is PlpD of Pseudomonas aeruginosa , an opportunistic human pathogen. The PlpD passenger is a homodimer with phospholipase A1 (PLA1) activity. Based on sequencing data, PlpD-like proteins are present in many bacterial species. We characterized the enzymatic activity, specific lipid binding and oligomeric status of PlpD homologs from Aeromonas hydrophila (a fish pathogen), Burkholderia pseudomallei (a human pathogen) and Ralstonia solanacearum (a plant pathogen) and compared these with PlpD. We demonstrate that recombinant type 5d-secreted patatin domains have lipase activity and form dimers or higher-order oligomers. However, dimerization is not necessary for lipase activity; in fact, by making monomeric variants of PlpD, we show that enzymatic activity slightly increases while protein stability decreases. The lipases from the intracellular pathogens A. hydrophila and B. pseudomallei display PLA2 activity in addition to PLA1 activity. Although the type 5d-secreted lipases from the animal pathogens bound to intracellular lipid targets, phosphatidylserine and phosphatidylinositol phosphates, hydrolysis of these lipids could only be observed for FplA of Fusobacterium nucleatum . Yet, we noted a correlation between high lipase activity in type 5d autotransporters and intracellular lifestyle. We hypothesize that type 5d phospholipases are intracellularly active and function in modulation of host cell signaling events

PT symmetry, Cartan decompositions, Lie triple systems and Krein space related Clifford algebras

Gauged PT quantum mechanics (PTQM) and corresponding Krein space setups are studied. For models with constant non-Abelian gauge potentials and extended parity inversions compact and noncompact Lie group components are analyzed via Cartan decompositions. A Lie triple structure is found and an interpretation as PT-symmetrically generalized Jaynes-Cummings model is possible with close relation to recently studied cavity QED setups with transmon states in multilevel artificial atoms. For models with Abelian gauge potentials a hidden Clifford algebra structure is found and used to obtain the fundamental symmetry of Krein space related J-selfadjoint extensions for PTQM setups with ultra-localized potentials.Comment: 11 page