Marine Litter : Technical Recommendations for the Implementation of MSFD Requirements

As a follow up to the Commission Decision on criteria and methodological standards on good environmental status (GES) of marine waters (Commission Decision 2010/477/EU), the Marine Directors requested Directorate General Environment in 2010 to establish a technical subgroup under the Working Group on GES in relation to the Marine Strategy Framework Directive 2008/56/EC (WG GES) for further development of Descriptor 10 Marine Litter and Descriptor 11 Noise/Energy. This report compiles the recommendations regarding Descriptor 10, Marine Litter. The implementation of provisons under MSFD Descriptor 10 as described in the Commission Decision 2010/477/EU is in its first step depending on the availability of appropriate monitoring tools.The group has investigated the monitoring approaches for marine litter and provides a set of monitoring tools which can be employed for that purpose.There are gaps in the regional applicability and differences in the maturity of some tools. There is need for further harmonization and collaborative activities in order to allow EU MS the future reporting of environmental trends and thus the verification of measures against marine litter.JRC.H.5-Rural, water and ecosystem resource

Nucleic acid and protein extraction from electropermeabilized E. coli cells on a microfluidic chip

Due to the extensive use of nucleic acid and protein analysis of bacterial samples, there is a need for simple and rapid extraction protocols for both plasmid DNA and RNA molecules as well as reporter proteins like the green fluorescent protein (GFP). In this report, an electropermeability technique has been developed which is based on exposing E. coli cells to low voltages to allow extraction of nucleic acids and proteins. The flow-through electropermeability chip used consists of a microfluidic channel with integrated gold electrodes that promote cell envelope channel formation at low applied voltages. This will allow small biomolecules with diameters less than 30 A to rapidly diffuse from the permeabilized cells to the surrounding solution. By controlling the applied voltage, partial and transient to complete cell opening can be obtained. By using DC voltages below 0.5 V, cell lysis can be avoided and the transiently formed pores can be closed again and the cells survive. This method has been used to extract RNA and GFP molecules under conditions of electropermeability. Plasmid DNA could be recovered when the applied voltage was increased to 2 V, thus causing complete cell lysis

An experimental and computational fluid dynamics study of the influence of fluid mixing and fluid stress on DNA purification

Interest in the field of pure DNA manufacture has been driven in recent years by the explosion of research into gene therapy. Gene therapy technology offers a new paradigm for treating human diseases where defective cells are transformed with gene vectors capable of expressing therapeutic protein. Administration is often via direct injection of naked or lipid-coated plasmid DNA. Plasmid for gene therapy is usually produced in Escherichia coli. The challenge in manufacturing plasmid is primarily the removal of impurities like proteins, lipids, lipopolysaccharides, RNA, non-supercoiled plasmid variants, and host chromosomal DNA. Long chain polymers, such as DNA, are uniquely prone to chain scission at moderate to high fluid stresses that commonly occur in biotechnology equipment. Stress-induced degradation of both plasmid DNA and host chromosomal DNA must be minimised to optimise plasmid yield and purity. Such degradation plays a critical role during alkaline lysis, a key step in DNA isolation. The effect of lysis reagent on DNA stability, the required level of fluid mixing, the effect of the resultant fluid stresses on DNA degradation, and the effect of DNA fragmentation on subsequent downstream purification performance are all poorly understood. This thesis sets out to characterise the effect of lysis reagent concentration on DNA so as to determine the required level of fluid mixing during alkaline lysis, to characterise the effect of the resultant fluid stress on DNA degradation and to determine the effects of stress-induced degradation on downstream processing. The following paragraphs outline the key finding of the thesis, which together provide a framework for the design of a robust lysis process. Two novel HPLC-based procedures were developed, based on polyethylenimine and quaternary amine anion exchange chromatography resins, capable of simultaneously measuring supercoiled plasmid DNA and chromosomal DNA in process samples, in addition the form of the chromosomal DNA. Experiments using E. coli cells containing 6 kb to 116 kb plasmids showed that cell lysate should be maintained below 0.13 + 0.03 M NaOH to prevent irreversible denaturation of supercoiled plasmids and above 0.08 M NaOH to ensure complete conversion of chromosomal DNA to single-stranded form. Conversion of chromosomal DNA to single-stranded form was shown not to significantly affect its removal during alkaline lysis, but was advantageous for subsequent purification. Complete conversion of chromosomal DNA to single-stranded form enabled complete removal by a variety of inexpensive and scaleable purification methods, significantly reducing the cost of plasmid DNA manufacture. Denaturation-renaturation of DNA, either during alkaline lysis or further downstream, was shown to be an effective method of removing non-supercoiled plasmid variants.The level of mixing required is highly dependent on the sodium hydroxide (NaOH) concentration in the lysis buffer. More highly concentrated lysis buffer reduced the overall lysate volume, but rapid mixing was essential to avoid irreversible supercoiled plasmid degradation. Mixing tanks provided adequate mixing only at low NaOH concentrations. Opposed jets provided excellent mixing characteristics for lysis buffer addition, and concentrated NaOH could be used, significantly reducing the volume increase over alkaline lysis. Opposed jets provided a suitable method for denaturing residual double-stranded chromosomal DNA downstream of alkaline lysis. Hence, inexpensive methods for singlestranded DNA removal could be utilised to remove all residual chromosomal DNA. Computational fluid dynamics (CFD) simulations were used to develop appropriate scaling rules for opposed jets, and the CFD predictions were verified against published experimental data. Capillary shear degradation studies with pure solutions of 6kb to 116 kb plasmids and chromosomal DNA, determined that DNA degraded at capillary entrances, not internally. Large plasmids degraded at significantly lower fluid flow rates than small plasmids. CFD simulations were used to determine fluid flow properties (turbulent energy dissipation rates, shear stresses, elongational stresses and pressure drops) at the entrance to, and within, capillaries and to correlate breakage of chromosomal and plasmid DNA with fluid flow parameters. Results indicated that elongational fluid stresses caused significantly more DNA degradation than shear stresses. An assay to monitor plasmid degradation in dilute solutions was developed using Picogreen dye, enabling different size plasmids to be used as probes for fluid stress-induced degradation in large-scale industrial equipment. Results showed that fluid stresses during alkaline lysis led to chromosomal DNA fragmentation. Despite causing chromosomal fragmentation, it was shown that fluid stresses during lysis did not significantly increase chromosomal contamination in cell lysates; chromosomal DNA removal over alkaline lysis/neutralisation not being a strong function of chromosomal DNA size. High levels of fluid stress during the neutralisation step were also shown not to increase chromosomal DNA contamination. The effects of chromosomal DNA fragment size on its removal in different downstream purification steps demonstrated which steps were sensitive to DNA size, enabling better selection of downstream unit operations based on DNA fragmentation upstream

An experimental and computational fluid dynamics study of the influence of fluid mixing and fluid shear on DNA during cell lysis

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Degradation of Supercoiled Plasmid DNA Within a Capillary Device

Supercoiled plasmid DNA is susceptible to fluid stress in large-scale manufacturing processes. A capillary device was used to generate controlled shear conditions and the effects of different stresses on plasmid DNA structure were investigated. Computational fluid dynamics (CFD) analysis was employed to characterize the flow environment in the capillary device and different analytical techniques were used to quantify the DNA breakage. It was found that the degradation of plasmid DNA occurred at the entrance of the capillary and that the shear stress within the capillary did not affect the DNA structure. The degradation rate of plasmids was well correlated with the average elongational strain rate or the pressure drop at the entrance region. The conclusion may also be drawn that laminar shear stress does not play a significant role in plasmid DNA degradation.F.J. Meacle, H. Zhang, I. Papantoniou, J.M. Ward, N.J. Titchener-Hooker and M. Hoar

Separation of genomic DNA, RNA, and open circular plasmid DNA from supercoiled plasmid DNA by combining denaturation, selective renaturation and aqueous two-phase extraction

In the current study we developed a process for the capture of pDNA exploiting the ability of aqueous two-phase systems to differentiate between different forms of DNA. In these systems scpDNA exhibits a near quantitative partitioning in the salt-rich bottom phase. The successive recovery from the salt rich bottom phase is accomplished by a novel membrane step. The polish operation to meet final purity demands is again based on a system exploiting a combination of the denaturation of the nucleic acids present, specific renaturation of scpDNA, and an ATP system able to differentiate between the renatured scpDNA and the denatured contaminants such as ocpDNA and genomic host DNA. This polish step thus allows a rapid and efficient separation of scpDNA from contaminating nucleic acids which up to date otherwise only can be accomplished with much more cumbersome chromatographic methods. In a benchmark comparison, it could be shown that the newly developed process exhibits a comparable yield to an industrial standard process while at the same time showing superior performance in terms of purity and process time. Additionally it could be shown that the developed polish procedure can be applied as a standalone module to support already existing processes