Fluorescence Microscopy and Gold Nanoparticles. View on pubs. Re-engineering of an E. The Escherichia coli phosphoglucose isomerase pgi mutant strain GALG20 was developed previously from wildtype K12 strain MG for increased plasmid yield. Elementary mode analysis EMA was then performed to compare the phenotypic spaces of both the strains and to check the effect of the pgi deletion on flux efficiency of each metabolic reaction.
View on doi. Colorimetric detection of D-dimer in a paper-based immunodetection device more.
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A fluorescent DNA probe was used to assess i the hybridization with complementary A fluorescent DNA probe was used to assess i the hybridization with complementary biotin-labeled target, ii the complexation of the resulting hybrid and an anti-biotin antibody and iii the binding of the latter complex to a ZZ-CBM fusion protein that combines small synthetic IgG Fc-binding Z domains with a carbohydrate binding module CBM. These binding interactions were monitored by exposing the fluorescent DNA probe to different amounts and combinations of the other molecules in solution. Through the analysis of FCS autocorrelation curves, an association constant Ka of 2.
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The FCS-derived information obtained is useful to gain insight into molecular interactions involved in diagnostic assays. Materials with new and improved functionalities can be obtained by modifying cellulose with gold nanoparticles AuNPs via the in situ reduction of a gold precursor or the deposition or covalent immobilization of pre-synthesized AuNPs Materials with new and improved functionalities can be obtained by modifying cellulose with gold nanoparticles AuNPs via the in situ reduction of a gold precursor or the deposition or covalent immobilization of pre-synthesized AuNPs.
Here, we present an alternative biomolecular recognition approach to functionalize cellulose with biotin-AuNPs that relies on a complex of 2 recognition elements: a ZZ-CBM3 fusion that combines a carbohydrate-binding module CBM with the ZZ fragment of the staphylococcal protein A and an anti-biotin antibody. Paper and cellulose microparticles with AuNPs immobilized via the ZZ-CBM3:anti-biotin IgG supramolecular complex displayed an intense red color, whereas essentially no color was detected when AuNPs were deposited over the unmodified materials.
Scanning electron microscopy analysis revealed a homogeneous distribution of AuNPs when immobilized via ZZ-CBM3:anti-biotin IgG complexes and aggregation of AuNPs when deposited over paper, suggesting that color differences are due to interparticle plasmon coupling effects. The approach could be used to functionalize paper substrates and cellulose nanocrystals with AuNPs. More important, however, is the fact that the occurrence of a biomolecular recognition event between the CBM-immobilized antibody and its specific, AuNP-conjugated antigen is signaled by red color.
Deconstruction of cellulose, the most abundant plant cell wall polysaccharide, requires the cooperative activity of a large repertoire of microbial enzymes.
Modular cellulases contain non-catalytic type A carbohydrate-binding modules Modular cellulases contain non-catalytic type A carbohydrate-binding modules CBMs that specifically bind to the crystalline regions of cellulose, thus promoting enzyme efficacy through proximity and targeting effects. Although type A CBMs play a critical role in cellulose recycling, their mechanism of action remains poorly understood. Here we produced a library of recombinant CBMs representative of the known diversity of type A modules. The binding properties of 40 CBMs, in fusion with an N-terminal GFP domain, revealed that type A CBMs possess the ability to recognize different crystalline forms of cellulose and chitin over a wide range of temperatures, pH levels, and ionic strengths.
A Spirochaeta thermophila CBM64, in particular, displayed plasticity in its capacity to bind both crystalline and soluble carbohydrates under a wide range of extreme conditions. The structure of S. Significantly, two highly conserved asparagine side chains, each one located between two tryptophan residues, are critical to insoluble and soluble glucan recognition but not to bind xyloglucan.
Thus, CBM64 compact structure and its extended and versatile ligand interacting platform illustrate how type A CBMs target their appended plant cell wall-degrading enzymes to a diversity of recalcitrant carbohydrates under a wide range of environmental conditions. View on dx. A process for supercoiled plasmid DNA purification based on multimodal chromatography more. Non-viral gene therapy and DNA vaccination currently hold great potential for treatment and prevention of genetic and acquired diseases.
The large-scale manufacturing of plasmid DNA pDNA vectors, and the downstream processing in particular, is one of the key aspects of process development required to move non-viral gene therapy to the clinic. To address the problematic isolation and purification of pDNA molecules , an alternative and cost effective process based on multimodal chromatography was developed. The process involves E. The process was reproducible and performed similarly with differently sized plasmids bp, bp and 10, bp. Separation of plasmid DNA topoisomers by multimodal chromatography more. The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases.
Here, we describe the separation of supercoiled sc and open circular oc topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid.
The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample. Development of a nicking endonuclease-assisted method for the purification of minicircles more. Minicircle MC DNA vectors are able to generate a high-level transgene expression in vivo, which is superior to the one afforded by conventional plasmids.
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MC vectors are produced by replicating a parental plasmid PP and promoting its MC vectors are produced by replicating a parental plasmid PP and promoting its recombination in Escherichia coli. This generates a MC with the expression cassette, and a miniplasmid MP with the replication segment. Unfortunately, wider use of MC vectors is hampered by difficulties in isolating the target MCs from their MP counterpart. In this proof-of-concept study, a reproducible process is described to improve the purification of supercoiled sc MCs that combines an in vitro enzymatic relaxation of sc MP impurities with topoisomer separation and RNA clearance by hydrophobic interaction chromatography HIC step.
At the early stage of vector design, a site for the nicking endonuclease Nb.
A process was then established that involves E. Next, an in vitro digestion step was carried out with Nb. As a result, sc MPs and non-recombined PPs were converted into the corresponding open circular oc forms whereas sc MCs remain unaffected. On the basis of agarose gel electrophoresis analysis, the sc MC-containing fractions were determined to be virtually free from nucleic acid impurities.
More Info: Rosa, A. Publication Name: Analytical Chemistry, 86 Biosensors , Molecular Diagnostics , and Paper Microfluidics. The topography and wettability of the underside of English weed Oxalis pes-caprae leaves and of their biomimetic replicas are investigated.
Polyvinyl siloxane molds were cast from the leaves and then filled with an epoxy pre-polymer to Polyvinyl siloxane molds were cast from the leaves and then filled with an epoxy pre-polymer to produce replicas. The particular topographical structures of leaves and replicas were evaluated by optical microscopy and Scanning Electron Microscopy SEM analysis. The static wettability of leaves and replicas was assessed by contact angle measurements, while the dynamic wettability was characterized by estimating contact angle hysteresis and studying the dynamic behavior of impacting water droplets.
A smooth glass slip and its replica were used as control surfaces. Also, trichomes in the original leaves could not be accurately reproduced due to their flexibility and fragility. Differences in wetting behavior were also evident from droplet impact experiments, with rebound regimes prevailing in the original leaves and regimes characterized by higher adhesion and larger dissipation predominating in the replicas. Nevertheless, the morphological features of the leaf transferred to the replica were sufficient to promote a clear hydrophobic behavior of the replica when compared with the smooth epoxy reference surface.
Structural instability of plasmid biopharmaceuticals: challenges and implications more. The global increase in the number of applications involving therapeutic plasmid DNA pDNA is creating a need for large amounts of highly stable and purified molecules. One of the main obstacles during the developmental stages of a new This review focuses on major instability determinants in pDNA.
Their elimination could be considered an important step towards the design of safer and more efficient plasmid molecules. Particular emphasis is given to mutations triggered by the presence of repeated sequences, instability events occurring during plasmid intracellular routing, instability mediated by insertion sequences and host genome integration. Rational engineering of Escherichia coli strains for plasmid biopharmaceutical manufacturing more. Prather , and Duarte Miguel Prazeres.
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Plasmid Biopharmaceuticals more. Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its mission to produce pharmaceutical proteins, antibodies, vaccines, industrial Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its mission to produce pharmaceutical proteins, antibodies, vaccines, industrial enzymes, and molecular diagnostics, to name a few key products.
Furthermore, plasmids have gradually stepped up in the past 20 years as useful biopharmaceuticals in the context of gene therapy and DNA vaccination interventions. This review provides a concise coverage of the scientific progress that has been made since the emergence of what are called today plasmid biopharmaceuticals.
The most relevant topics are discussed to provide researchers with an updated overview of the field. A brief outline of the initial breakthroughs and innovations is followed by a discussion of the motivation behind the medical uses of plasmids in the context of therapeutic and prophylactic interventions.
http://ehyxapolyq.tk The molecular characteristics and rationale underlying the design of plasmid vectors as gene transfer agents are described and a description of the most important methods used to deliver plasmid biopharmaceuticals in vivo gene gun, electroporation, cationic lipids and polymers, and micro- and nanoparticles is provided. The major safety issues integration and autoimmunity surrounding the use of plasmid biopharmaceuticals is discussed next.
Aspects related to the large-scale manufacturing are also covered, and reference is made to the plasmid products that have received marketing authorization as of today. More Info: Duarte Miguel F. Prazeres, Gabriel A. View on asmscience. De novo creation of MGderived E.