Expression and purification of an adenylation domain from a eukaryotic nonribosomal peptide synthetase: Using structural genomics tools for a challenging target

Nonribosomal peptide synthetases (NRPSs) are large multimodular and multidomain enzymes that are involved in synthesising an array of molecules that are important in human and animal health. NRPSs are found in both bacteria and fungi but most of the research to date has focused on the bacterial enzymes. This is largely due to the technical challenges in producing active fungal NRPSs, which stem from their large size and multidomain nature. In order to target fungal NRPS domains for biochemical and structural characterisation, we tackled this challenge by using the cloning and expression tools of structural genomics to screen the many variables that can influence the expression and purification of proteins. Using these tools we have screened 32 constructs containing 16 different fungal NRPS domains or domain combinations for expression and solubility. Two of these yielded soluble protein with one, the third adenylation domain of the SidN NRPS (SidNA3) from the grass endophyte Neotyphodium lolii, being tractable for purification using Ni-affinity resin. The initial purified protein exhibited poor solution behaviour but optimisation of the expression construct and the buffer conditions used for purification, resulted in stable recombinant protein suitable for biochemical characterisation, crystallisation and structure determination

Limits to ion energy control in high density glow discharges: Measurement of absolute metastable ion concentrations

Unprecedented demands for uniformity, throughput, anisotropy, and damage control in submicron pattern transfer are spurring development of new, low pressure, high charge density plasma reactors. Wafer biasing, independent of plasma production in these new systems is intended to provide improved ion flux and energy control so that selectivity can be optimized and damage can be minimized. However, as we show here, an inherent property of such discharges is the generation of significant densities of excited, metastable ionic states that can bombard workpiece surfaces with higher translational and internal energy. Absolute metastable ion densities are measured using the technique of self-absorption, while the corresponding velocity distributions and density scaling with pressure and electron density are measured using laser-induced fluorescence. For a low pressure, helicon-wave excited plasma, the metastable ion flux is at least 24% of the total ion flux to device surfaces. Because the metastable ion density scales roughly as the reciprocal of the pressure and as the square of the electron density, the metastable flux is largest in low pressure, high charge density plasmas. This metastable ion energy flux effectively limits ion energy and flux control in these plasma reactors, but the consequences for etching and deposition of thin films depend on the material system and remain an open question

Phenomenology of the Baryon Resonance 70-plet at Large N_c

We examine the multiplet structure and decay channels of baryon resonances in the large N_c QCD generalization of the N_c = 3 SU(6) spin-flavor 70. We show that this ``70'', while a construct of large N_c quark models, actually consists of five model-independent irreducible spin-flavor multiplets in the large N_c limit. The preferred decay modes for these resonances fundamentally depend upon which of the five multiplets to which the resonance belongs. For example, there exists an SU(3) ``8'' of resonances that is eta-philic and pi-phobic, and an ``8'' that is the reverse. Moreover, resonances with a strong SU(3) ``1'' component prefer to decay via a K-bar rather than via a pi. Remarkably, available data appears to bear out these conclusions.Comment: 26 pages, ReVTe

Fermions in 3D Optical Lattices: Cooling Protocol to Obtain Antiferromagnetism

A major challenge in realizing antiferromagnetic (AF) and superfluid phases in optical lattices is the ability to cool fermions. We determine the equation of state for the 3D repulsive Fermi-Hubbard model as a function of the chemical potential, temperature and repulsion using unbiased determinantal quantum Monte Carlo methods, and we then use the local density approximation to model a harmonic trap. We show that increasing repulsion leads to cooling, but only in a trap, due to the redistribution of entropy from the center to the metallic wings. Thus, even when the average entropy per particle is larger than that required for antiferromagnetism in the homogeneous system, the trap enables the formation of an AF Mott phase.Comment: 4 pages; 5 figures; also see supplementary material in 2 pages with 1 figur

Measurement of the Hyperfine Structure and Isotope Shifts of the 3s23p2 3P2 to 3s3p3 3Do3 Transition in Silicon

The hyperfine structure and isotope shifts of the 3s23p2 3P2 to 3s3p3 3Do3 transition in silicon have been measured. The transition at 221.7 nm was studied by laser induced fluorescence in an atomic Si beam. For 29Si, the hyperfine A constant for the 3s23p2 3P2 level was determined to be -160.1+-1.3 MHz (1 sigma error), and the A constant for the 3s3p3 3Do3 level is -532.9+-0.6 MHz. This is the first time that these constants were measured. The isotope shifts (relative to the abundant isotope 28Si) of the transition were determined to be 1753.3+-1.1 MHz for 29Si and 3359.9+-0.6 MHz for 30Si. This is an improvement by about two orders of magnitude over a previous measurement. From these results we are able to predict the hyperfine structure and isotope shift of the radioactive 31Si atom, which is of interest in building a scalable quantum computer

Stellar Populations and Star Cluster Formation in Interacting Galaxies with the Advanced Camera for Surveys

Pixel-by-pixel colour-magnitude and colour-colour diagrams - based on a subset of the Hubble Space Telescope Advanced Camera for Surveys Early Release Observations - provide a powerful technique to explore and deduce the star and star cluster formation histories of the Mice and the Tadpole interacting galaxies. In each interacting system we find some 40 bright young star clusters (20 <= F606W (mag) <= 25, with a characteristic mass of ~3 x 10^6 Msun), which are spatially coincident with blue regions of active star formation in their tidal tails and spiral arms. We estimate that the main events triggering the formation of these clusters occurred ~(1.5-2.0) x 10^8 yr ago. We show that star cluster formation is a major mode of star formation in galaxy interactions, with >= 35% of the active star formation in encounters occurring in star clusters. This is the first time that young star clusters have been detected along the tidal tails in interacting galaxies. The tidal tail of the Tadpole system is dominated by blue star forming regions, which occupy some 60% of the total area covered by the tail and contribute ~70% of the total flux in the F475W filter (decreasing to ~40% in F814W). The remaining pixels in the tail have colours consistent with those of the main disk. The tidally triggered burst of star formation in the Mice is of similar strength in both interacting galaxies, but it has affected only relatively small, spatially coherent areas.Comment: 23 pages in preprint form, 6 (encapsulated) postscript figures; accepted for publication in New Astronomy; ALL figures (even the grey-scale ones) need to be printed on a colour printer style files included; for full-resolution paper, see http://www.ast.cam.ac.uk/STELLARPOPS/ACSpaper