Dual affinity method for plasmid DNA purification in aqueous two-phase systems

The DNA binding fusion protein, LacI-His6-GFP, together with the conjugate PEG-IDA-Cu(II) (10 kDa) was evaluated as a dual affinity system for the pUC19 plasmid extraction from an alkaline bacterial cell lysate in poly(ethylene glycol) (PEG)/dextran (DEX) aqueous two-phase systems (ATPS). In a PEG 600-DEX 40 ATPS containing 0.273 nmol of LacI fusion protein and 0.14% (w/w) of the functionalised PEG-IDA-Cu(II), more than 72% of the plasmid DNA partitioned to the PEG phase, without RNA or genomic DNA contamination as evaluated by agarose gel electrophoresis. In a second extraction stage, the elution of pDNA from the LacI binding complex proved difficult using either dextran or phosphate buffer as second phase, though more than 75% of the overall protein was removed in both systems. A maximum recovery of approximately 27% of the pCU19 plasmid was achieved using the PEG-dextran system as a second extraction system, with 80-90% of pDNA partitioning to the bottom phase. This represents about 7.4 microg of pDNA extracted per 1 mL of pUC19 desalted lysate.

Affinity partitioning of plasmid DNA with a zinc finger protein

The affinity isolation of pre-purified plasmid DNA (pDNA) from model buffer solutions using native and poly(ethylene glycol) (PEG) derivatized zinc finger–GST (Glutathione-S-Transferase) fusion protein was examined in PEG–dextran (DEX) aqueous two-phase systems (ATPSs). In the absence of pDNA, partitioning of unbound PEGylated fusion protein into the PEG-rich phase was confirmed with 97.5% of the PEGylated fusion protein being detected in the PEG phase of a PEG 600–DEX 40 ATPS. This represents a 1322-fold increase in the protein partition coefficient in comparison to the non-PEGylated protein (Kc = 0.013). In the presence of pDNA containing a specific oligonucleotide recognition sequence, the zinc finger moiety of the PEGylated fusion protein bound to the plasmid and steered the complex to the PEG-rich phase. An increase in the proportion of pDNA that partitioned to the PEG-rich phase was observed as the concentration of PEGylated fusion protein was increased. Partitioning of the bound complex occurred to such an extent that no DNA was detected by the picogreen assay in the dextran phase. It was also possible to partition pDNA using a non-PEGylated (native) zinc finger–GST fusion protein in a PEG 1000–DEX 500 ATPS. In this case the native ligand accumulated mainly in the PEG phase. These results indicate good prospects for the design of new plasmid DNA purification methods using fusion proteins as affinity ligands.

Two-phase flow analogy as an effective boundary condition for modelling liquids at atomistic resolution

A hybrid Molecular Dynamics/Fluctuating Hydrodynamics framework based on the analogy with two-phase hydrodynamics has been extended to dynamically tracking the feature of interest at all-atom resolution. In the model, the hydrodynamics description is used as an effective boundary condition to close the molecular dynamics solution without resorting to standard periodic boundary conditions. The approach is implemented in a popular Molecular Dynamics package GROMACS and results for two biomolecular systems are reported. A small peptide dialanine and a complete capsid of a virus porcine circovirus 2 in water are considered and shown to reproduce the structural and dynamic properties compared to those obtained in theory, purely atomistic simulations, and experiment.

Development of two forecasting systems and the design of hard copy information displays

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY AND INFORMATION SERVICES WITH PRIOR ARRANGEMENT

Evaluation of new processes for the preparation of iron by gas/solid phase systems

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY AND INFORMATION SERVICES WITH PRIOR ARRANGEMENT

A meshless method for an inverse two-phase one-dimensional nonlinear Stefan problem

We extend a meshless method of fundamental solutions recently proposed by the authors for the one-dimensional two-phase inverse linear Stefan problem, to the nonlinear case. In this latter situation the free surface is also considered unknown which is more realistic from the practical point of view. Building on the earlier work, the solution is approximated in each phase by a linear combination of fundamental solutions to the heat equation. The implementation and analysis are more complicated in the present situation since one needs to deal with a nonlinear minimization problem to identify the free surface. Furthermore, the inverse problem is ill-posed since small errors in the input measured data can cause large deviations in the desired solution. Therefore, regularization needs to be incorporated in the objective function which is minimized in order to obtain a stable solution. Numerical results are presented and discussed. © 2014 IMACS.

Experiments for CFD-modelling of cooling water and Insulation debris two-phase flow phenomena during loss of coolant accidents

The knowledge of insulation debris generation and transport gains in importance regarding reactor safety research for PWR and BWR. The insulation debris released near the break consists of a mixture of very different fibres and particles concerning size, shape, consistence and other properties. Some fraction of the released insulation debris will be transported into the reactor sump where it may affect emergency core cooling. Experiments are performed to blast original samples of mineral wool insulation material by steam under original thermal-hydraulic break conditions of BWR. The gained fragments are used as initial specimen for further experiments at acrylic glass test facilities. The quasi ID-sinking behaviour of the insulation fragments are investigated in a water column by optical high speed video techniques and methods of image processing. Drag properties are derived from the measured sinking velocities of the fibres and observed geometric parameters for an adequate CFD modelling. In the test rig "Ring line-II" the influence of the insulation material on the head loss is investigated for debris loaded strainers. Correlations from the filter bed theory are adapted with experimental results and are used to model the flow resistance depending on particle load, filter bed porosity and parameters of the coolant flow. This concept also enables the simulation of a particular blocked strainer with CFDcodes. During the ongoing work further results of separate effect and integral experiments and the application and validation of the CFD-models for integral test facilities and original containment sump conditions are expected.

Comparative performance between two photogrammetric systems and a reference laser tracker network for large-volume industrial measurement

This paper determines the capability of two photogrammetric systems in terms of their measurement uncertainty in an industrial context. The first system – V-STARS inca3 from Geodetic Systems Inc. – is a commercially available measurement solution. The second system comprises an off-the-shelf Nikon D700 digital camera fitted with a 28 mm Nikkor lens and the research-based Vision Measurement Software (VMS). The uncertainty estimate of these two systems is determined with reference to a calibrated constellation of points determined by a Leica AT401 laser tracker. The calibrated points have an average associated standard uncertainty of 12·4 μm, spanning a maximum distance of approximately 14·5 m. Subsequently, the two systems’ uncertainty was determined. V-STARS inca3 had an estimated standard uncertainty of 43·1 μm, thus outperforming its manufacturer's specification; the D700/VMS combination achieved a standard uncertainty of 187 μm.

The effect of flow regimes in the distillation of systems containing two liquid phases

Single phase solutions containing three components have been observed to exhibit foaminess near a single to two liquid phase boundary. It was seen, in a sintered plate column under mass transfer conditions, that distillation systems where the liquid appeared as one phase in one part of a column and two phases in another part, exhibited foaminess when the liquid concentration was near the one phase to two phase boundary. Various ternary systems have been studied in a 50 plate. 30mm i.d. Oldershaw column and it was observed that severe foaming occurred in the middle section of the column near the one liquid phase to two liquid phase boundary and no foaming occurred at the end of the column where liquid was either one phase or two phase. This is known as Ross type foam. Mass transfer experiments with Ross type ternary systems have been carried out in a perspex simulator with small and large hole diameter trays. It was observed that by removal of the more volatile component, Ross type foam did not build up on the tray. Severe entrainment of liquid was observed in all cases leading to a 'dry' tray, even with a low free area small diameter hole tray which was expected to produce a bubbly mixture. Entrainment was more severe for high gas superficial velocities and large hole diameters. This behaviour is quite different from the build up of foam observed when one liquid phase/two liquid phase Ross systems were contacted with air above a small sintered disc or with vapour in an Oldershaw distillation column. This observation explains why distillation columns processing mixtures which change from one liquid phase to two liquid phases (or vice versa) must be severely derated to avoid flooding. Single liquid phase holdups at the spray to bubbly transition were measured using a perspex simulator similar to that of Porter & Wong (17). i.e. with no liquid cross flow. A light transmission technique was used to measure the transition from spray regime to bubbly regime. The effect of tray thickness and the ratio of hole diameter to tray thickness on the transition was evaluated using trays of the same hole diameter and free area but having thickness of 2.38 mm, 4 mm, and 6.35 mm. The liquid holdup at the transition was less with the thin metal trays. This result may be interpreted by the theory of Lockett (101), which predicts the transition liquid holdup in terms of the angle of the gas iet leaving the holes in the sieve plate. All the existing correlations have been compared and none were found to be satisfactory and these correlations have been modified in view of the experimental results obtained. A new correlation has been proposed which takes into account the effect of the hole diameter to tray thickness ratio on the transition and good agreement was obtained between the experimental results and the correlated values of the liquid holdup at the transition. Results have been obtained for two immiscible liquids [kerosene and water] on trays to determine whether foaming can be eliminated by operating in the spray regime. Kerosene was added to a fixed volume of water or water was added to a fixed volume of kerosene. In both cases, there was a transition from spray to bubbly. In the water fixed system. the liquid holdup at the transition was slightly less than the pure kerosene system. Whilst for the kerosene fixed system, the transition occurred at much lower liquid holdups. Trends In the results were similar to those for single liquid phase. New correlations have been proposed for the two cases. It has been found that Ross type foams, observed in a sintered plate column and in the Oldershaw column can be eliminated by either carrying out the separation in a packed column or by the addition of defoaming additives.

Studies in three phase gas-liquid-solid fluidised systems

The work is a logical continuation of research started at Aston some years ago when studies were conducted on fermentations in bubble columns. The present work highlights typical design and operating problems that could arise in such systems as waste water, chemical, biochemical and petroleum operations involving three-phase, gas-liquid-solid fluidisation; such systems are in increasing use. It is believed that this is one of few studies concerned with `true' three-phase, gas-liquid-solid fluidised systems, and that this work will contribute significantly to closing some of the gaps in knowledge in this area. The research work was mainly experimentally based and involved studies of the hydrodynamic parameters, phase holdups (gas and solid), particle mixing and segregation, and phase flow dynamics (flow regime and circulation patterns). The studies have focused particularly on the solid behaviour and the influence of properties of solids present on the above parameters in three-phase, gas-liquid-solid fluidised systems containing single particle components and those containing binary and ternary mixtures of particles. All particles were near spherical in shape and two particle sizes and total concentration levels were used. Experiments were carried out in two- and three-dimensional bubble columns. Quantitative results are presented in graphical form and are supported by qualitative results from visual studies which are also shown as schematic diagrams and in photographic form. Gas and solid holdup results are compared for air-water containing single, binary and ternary component particle mixtures. It should be noted that the criteria for selection of the materials used are very important if true three-phase fluidisation is to be achieved: this is very evident when comparing the results with those in the literature. The fluid flow and circulation patterns observed were assessed for validation of the generally accepted patterns, and the author believes that the present work provides more accurate insight into the modelling of liquid circulation in bubble columns. The characteristic bubbly flow at low gas velocity in a two-phase system is suppressed in the three-phase system. The degree of mixing within the system is found to be dependent on flow regime, liquid circulation and the ratio of solid phase physical properties. Evidence of strong `trade-off' of properties is shown; the overall solid holdup is believed to be a major parameter influencing the gas holdup structure.