A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering

<p>Abstract</p> <p>Background</p> <p>The combined use of restriction enzymes with PCR has revolutionized molecular cloning, but is inherently restricted by the content of the manipulated DNA sequences. Uracil-excision based cloning is ligase and sequence independent and allows seamless fusion of multiple DNA sequences in simple one-tube reactions, with higher accuracy than overlapping PCR.</p> <p>Results</p> <p>Here, the addition of a highly efficient DNA polymerase and a low-background-, large-insertion- compatible site-directed mutagenesis protocol is described, largely expanding the versatility of uracil-excision DNA engineering.</p> <p>Conclusions</p> <p>The different uracil-excision based molecular tools that have been developed in an open-source fashion, constitute a comprehensive, yet simple and inexpensive toolkit for any need in molecular cloning.</p

svenska

&nbsp;The price of housing - on the pedagogical function of a public audit, on informal learning in the civil society, and on the possibility of expressing an opinion The article contains an analysis of parts of a formalized public audit of the proposed development of a local residential area in Sweden, involving i.a. the clearing of a small urban forest. The subject of the analysis is the comments received in the third out of three public reviews. The empirical findings are analyzed in order to understand and explain the popular involvement in the audit as well as the expressed frustration that none of the people's comments had any impact on the final project. The analysis sets out from Pierre Bourdieu's practice praxeology. Perspectives are also given to theories of/for informal learning in civil society, to Hannah Arendt's philosophy of the banality of evil (of bureaucracy), and to recent analyzes of developments in municipal housing policy, and of the social dynamics of the elite, its choice of education and place of residence. Finally, both the empirical theory of evaluations/evaluating practices and the sociology of representation developed by the author are implemented. The public audit and the associated informal learning in civil society is part of a democratic ideal that the people keep the regime in check through certain formalized practices. However, the analysis shows that the audit, an intricate system of evaluating practices, serves to hinder the influence of the people, and that the local politicians and bureaucrats tend to give priority to the socially dominant (mainly economic) interests rather than serve the people. Similarly, any investigation related to the audit has the function of hindering the production of systematic knowledge. Consequently, existing dominant interests are taken into account at the expense of any other. Not only the natural basis exploited. Even the people is a resource for exploitation, facilitated by the municipality

Outlining a theory of the social and symbolic function of evaluations of education

AbstractThe article presents the results of a research project focusing on evaluations of education as a part of a New Public Management in the area of education.The empirical material consists of:- 8 state-sanctioned evaluations of the formal training programs for the positions in a medical field- various texts on evaluations- various examples of Danish evaluation research.A field of producers of Danish evaluation research is constructed as part of a field of power: analogous to the analysed evaluations, Danish evaluation research forms a discourse legitimizing socially necessary administrative interventions. The evaluations and the evaluation research are constructed as parts of a mechanism performing and legitimizing a sorting to an existing social order. The theoretical starting point is from theories, primarily by Émile Durkheim, Pierre Bourdieu and Ulf P. Lundgren.Keywords: evaluation, evaluation of education, social reproduction, New Public Management, societies after the Modern, meritocrac

A versatile one-step CRISPR-Cas9 based approach to plasmid-curing

Abstract Background Plasmids are widely used and essential tools in molecular biology. However, plasmids often impose a metabolic burden and are only temporarily useful for genetic engineering, bio-sensing and characterization purposes. While numerous techniques for genetic manipulation exist, a universal tool enabling rapid removal of plasmids from bacterial cells is lacking. Results Based on replicon abundance and sequence conservation analysis, we show that the vast majority of bacterial cloning and expression vectors share sequence similarities that allow for broad CRISPR-Cas9 targeting. We have constructed a universal plasmid-curing system (pFREE) and developed a one-step protocol and PCR procedure that allow for identification of plasmid-free clones within 24 h. While the context of the targeted replicons affects efficiency, we obtained curing efficiencies between 40 and 100% for the plasmids most widely used for expression and engineering purposes. By virtue of the CRISPR-Cas9 targeting, our platform is highly expandable and can be applied in a broad host context. We exemplify the wide applicability of our system in Gram-negative bacteria by demonstrating the successful application in both Escherichia coli and the promising cell factory chassis Pseudomonas putida. Conclusion As a fast and freely available plasmid-curing system, targeting virtually all vectors used for cloning and expression purposes, we believe that pFREE has the potential to eliminate the need for individualized vector suicide solutions in molecular biology. We envision the application of pFREE to be especially useful in methodologies involving multiple plasmids, used sequentially or simultaneously, which are becoming increasingly popular for genome editing or combinatorial pathway engineering

Pierre Bourdieu

Abstract &nbsp; Med udgangspunkt i især habitus-begrebet og praktikteorien handler denne artikel om Pierre Bourdieus teorier og arbejde, den historie og samtid som betingede arbejdet, en teoretisk positionering af arbejdet, herunder den måde alt dette er blevet modtaget, ikke kun af dem der anser at arbejdet er frugtbart at arbejde med eller lade sig inspirere af, men desuden af dem som mener at arbejdet er misforstået, inkonsistent eller uanvendeligt af den ene eller anden grund. Overskrifterne i artiklen signalerer den måde hvorpå indholdet præsenteres: 1. Sociologiske konsekvenser af en biografi 2. Bourdieus position på det filosofiske og det sociologiske område 3. Forfatterskabet i korte træk 4. Den sociale verden og kundskaben om denne verden 4.a. Den spontane hverdagsbevidsthed og de hjemmelavede teorier 4.b. Videnskab som brud med den umiddelbare oplevelse 4.c Videnskabeliggørelsen af den praktiske sans? 4.d. Videnskaben som brud med videnskaben 4.e. Det sociale rum, det autonome felt, dispositioner og positioner, den symbolske vold og habitus 5. Staten og uddannelsessystemet: om symbolsk, økonomisk, social og kulturel kapital 6. Den relationistiske måde at konstruere teori går ud på at: 7. Bourdieu og hans kritikere 8. Bourdieu i Skandinavien 9. NøglebegreberAbstract &nbsp; Setting out from the notion of Habitus and the theory of practice of Pierre Bourdieu this article treats his theories and his work, the history and the contemporary society conditioning the work, a theoretical positioning of the work, including the reception hereof not only by those who find it fruitful to work with or being inspired by the work, but also by those who find that the work is a misunderstanding, inconsistent, or useless for whatever reason. The headings of the article signal the way the content is presented: 1. Sociological consequences of a biography 2. The position of Bourdieu in the philosophical and sociological domains 3. The works in short 4. The social world and the knowledge about this world 4. a. The spontaneous everyday conscience and the homemade theories 4.b. Science as a break with the immediate experience 4.c. Scientification of the practical sense? 4.d. Science as a break with science 4.e. The social space, the autonomous field, dispositions and positions, the symbolic violence, and habitus 5. The state and the educational system: on symbolic, economic, social and cultural capital 6. The relationistic way to construct a theory 7. Bourdieu and his critics 8. Bourdieu in Scandinavia 9. Key concept

Accurate DNA assembly and genome engineering with optimized uracil excision cloning

Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of <i>gfp</i> and a four-gene pathway that produces β-carotene to optimize assembly junctions and the uracil excision protocol. By combining uracil excision cloning with a genomic integration technology, we demonstrate that up to six DNA fragments can be assembled in a one-tube reaction for direct genome integration with high accuracy, greatly facilitating the advanced engineering of robust cell factories

Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes

BACKGROUND: We have developed a functional genomics approach based on expression cloning in Xenopus oocytes to identify plant transporter function. We utilized the full-length cDNA databases to generate a normalized library consisting of 239 full-length Arabidopsis thaliana transporter cDNAs. The genes were arranged into a 96-well format and optimized for expression in Xenopus oocytes by cloning each coding sequence into a Xenopus expression vector. RESULTS: Injection of 96 in vitro transcribed cRNAs from the library in pools of columns and rows into oocytes and subsequent screening for glucose uptake activity identified three glucose transporters. One of these, AtSTP13, had not previously been experimentally characterized. CONCLUSION: Expression of the library in Xenopus oocytes, combined with uptake assays, has great potential in assignment of plant transporter function and for identifying membrane transporters for the many plant metabolites where a transporter has not yet been identified