732 research outputs found
Isolation and Characterization of Metalloproteases with a Novel Domain Structure by Construction and Screening of Metagenomic Libraries
No full textSmall-insert metagenomic libraries from four samples were constructed by a topoisomerase-based and a T4 DNA ligase-based approach. Direct comparison of both approaches revealed that application of the topoisomerase-based method resulted in a higher number of insert-containing clones per mu g of environmental DNA used for cloning and a larger average insert size. Subsequently, the constructed libraries were partially screened for the presence of genes conferring proteolytic activity. The function-driven screen was based on the ability of the library-containing Escherichia coli clones to form halos on skim milk-containing agar plates. The screening of 80,000 E. coli clones yielded four positive clones. Two of the plasmids (pTW2 and pTW3) recovered from positive clones conferred strong proteolytic activity and were studied further. Analysis of the entire insert sequences of pTW2 (28,113 bp) and pTW3 (19,956 bp) suggested that the DNA fragments were derived from members of the genus Xanthomonas. Each of the plasmids harbored one gene (2,589 bp) encoding a metalloprotease (mprA, pTW2; mprB, pTW3). Sequence and biochemical analyses revealed that MprA and MprB are similar extracellular proteases belonging to the M4 family of metallopeptidases (thermolysin-like family). Both enzymes possessed a unique modular structure and consisted of four regions: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The architecture of the latter region, which was characterized by the presence of two prepeptidase C-terminal domains and one proprotein convertase P domain, is novel for bacterial metalloproteases. Studies with derivatives of MprA and MprB revealed that the C-terminal extension is not essential for protease activity. The optimum pH and temperature of both proteases were 8.0 and 65 degrees C, respectively, when casein was used as substrate
Complete Genome Sequence of the B-12-Producing Shimwellia blattae Strain DSM 4481, Isolated from a Cockroach
No full textHere we announce the complete genome sequence of the coenzyme B-12-producing enteric bacterium Shimwellia blattae (formerly Escherichia blattae). The genome consists of a single chromosome (4,158,636 bp). The genome size is smaller than that of most other enteric bacteria. Genome comparison revealed significant differences from the Escherichia coli genome
Tanja Dixon-Warren’s Story of Jane
No full textgardenimmigrantoriginalVancouverWorld War II1930’sBritai
Internationalisation Strategies for B2B Companies
No full textAuthor Tanja Maria Scherbaum, B.A.Masterarbeit Universität Linz 201
Internationalisation Strategies for B2B Companies
No full textAuthor Tanja Maria Scherbaum, B.A.Masterarbeit Universität Linz 201
Der Einfluss von Social Media Marketing von Luxus-Modemarken auf das Markenbewusstsein und die Kaufabsicht der Kunden : am Beispiel von Instagram
No full textAuthor Heinzl Tanja, BScAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMasterarbeit Universität Linz 2022Arbeit auf den öffentlichen PCs in den Bibliotheken der JKU+Medizin abrufba
Engagement dynamics In online workshops for future-making : a case study of an open foresight project
No full textAuthor Tanja Berndl BScMasterarbeit Johannes Kepler Universität Linz 2024Arbeit nach Ablauf der Sperre auf den öffentlichen PCs in den Bibliotheken der JKU+Medizin abrufba
Engagement dynamics In online workshops for future-making : a case study of an open foresight project
No full textAuthor Tanja Berndl BScMasterarbeit Johannes Kepler Universität Linz 2024Arbeit nach Ablauf der Sperre auf den öffentlichen PCs in den Bibliotheken der JKU+Medizin abrufba
Identification and characterization of genes and encoding gene products of metagenomic libraries conferring butanol dehydrogenase activity or proteolytic activity
No full textIn den vergangenen Jahren wurde es durch kultivierungsunabhängige metagenomische Analysen ermöglicht, die natürlich vorkommende mikrobielle Diversität als nahezu unerschöpfliche Ressource für neuartige Biokatalysatoren und Wirkstoffe zu erschließen. Dazu wird die gesamte mikrobielle DNA aus einer Standortprobe isoliert und direkt kloniert. Die Hinterlegung der auf diesem Weg erhaltenen genetischen Informationen erfolgt in komplexen Genbanken, so genannten Metagenombanken. Diese können in heterologen Wirten beliebig oft vermehrt, mit sequenzabhängigen oder auf biologischer Aktivität basierenden Verfahren analysiert und sowohl für die Anwendung als auch für die Forschung genutzt werden. Im Rahmen dieser Arbeit wurden verschiedene Metagenombanken konstruiert und aktivitätsbasierend auf Gene durchmustert, die Butanol-Dehydrogenase-Aktivität oder proteolytische Aktivität vermitteln. Die für die entsprechenden Aktivitäten verantwortlichen Gene wurden anschließend identifiziert und charakterisiert.Im Rahmen des Butanol-Dehydrogenase-Screenings konnten zwei positive Escherichia coli-Klone identifiziert werden, deren rekombinanten Plasmide (pTWB3 bzw. pTWB4) in der Lage waren, eine stabile NADPH-abhägige Butanol-Dehydrogenase-Aktivität zu vermitteln. Durch weitere Untersuchungen konnte bei pTWB4 das für die Aktivität verantwortliche Gen identifiziert werden. Die abgeleitete Aminosäuresequenz des Genprodukts zeigte keinerlei Ähnlichkeiten zu Proteinsequenzen von bekannten Alkohol-Dehydrogenasen oder Oxidoreduktasen, sondern wies große Übereinstimmungen zu der Proteinsequenz einer Oligoendopeptidase aus Bacillus licheniformis auf.Im Rahmen des Screenings nach proteolytischer Aktivität konnten fünf positive E. coli Klone identifiziert werden, von denen zwei näher charakterisiert wurden. Auf den Insertsequenzen der aus diesen Stämmen isolierten rekombinanten Plasmide, konnte jeweils ein Protease-Gen (mprA und mprB) identifiziert werden. Die Aminosäuresequenzen der beiden abgeleiteten Genprodukte zeigten Ähnlichkeiten zu einer Zink-abhängigen Metalloprotease aus Pseudoalteromonas sp. str. A28. Durch weitere Sequenzanalysen konnten MprA und MprB in die Familie M4 der Zink-abhängigen Metalloproteasen eingestuft werden. Proteasen dieser Familie zeichnen sich dadurch aus, dass es sich um sekretierte Enzyme handelt, welche als inaktive Vorläufer Proteine gebildet werden. Sie besitzen N-terminale und teilweise auch C-terminale Prosequenzen, denen unterschiedliche Funktionen bei der Reifung der Proteasen zugesprochen wird. Die Gene mprA und mprB sowie unterschiedliche Derivate mit Deletionen im N-terminalen bzw. C-terminalen Genbereichen wurden in E. coli BL21-Zellen produziert. Es konnte gezeigt werden, dass der N-terminale Genbereich im Gegensatz zum C-terminalen Genbereich essentiell für die proteolytische Aktivität der Proteasen ist. Ferner zeigten nur in den Kulturüberstand exportierte Enzyme Aktivität. Weiterhin erfolgte eine biochemische Charakterisierung der aktiven Proteasen und ihrer aktiven Derivate.The cultivation-independent assessment and exploitation of the vast microbial diversity by metagenomics offers an almost unlimited resource of new genes, gene products, and biosynthetic pathways for biotechnological and other purposes. The metagenomic approach is based on direct isolation of DNA from environmental samples and on cloning of the recovered DNA into vectors. For retrieval of novel products, the constructed so-called metagenomic libraries are then sequence-based or activity-based screened for genes encoding stable biocatalysts and other microbial products of interest. In this study, complex metagenomic libraries from different environments were constructed and screened for genes conferring butanol dehydrogenase activity or proteolytic activity. Subsequently, the genes and gene products that were responsible for the targeted activities were identified and characterized.The screening of the metagenomic libraries for butanol dehydrogenase activity yielded two positive Escherichia coli clones. The recombinant plasmids recovered from these clones (pTWB3 and pTWB4) conferred a stable and NADPH-dependent butanol dehydrogenase activity. Further characterization of pTWB4 resulted in the identification of the gene that is responsible for the activity. The amino acid sequence deduced from the identified gene showed no homology to known alcohol dehydrogenases or oxidoreductases but revealed significant similarities to an oligoendopeptidase from Bacillus licheniformis.The screening of the constructed metagenomic libraries for genes encoding proteolytic activity yielded five positive E. coli clones of which two were studied further. The insert sequences of the plasmids that were recovered from these clones harbored each a gene coding for a protease (mprA and mprB). The deduced amino acid sequences of MprA and MprB showed significant similarities to a zinc-dependent metalloprotease of Pseudoalteromonas sp. str. A28. Further analysis revealed that both enzymes belong to family M4 of the zinc-dependent metalloproteases. Members of this family are formed as inactive precursors and are secreted. In addition, M4 metalloproteases contain N-terminal and in some cases C-terminal pro-peptides, which play an important role during maturation of the proteins. The gene products of mprA, mprB, and derivatives of these genes that contained deletions in the N-terminal or C-terminal regions were produced in E.coli BL21 and analyzed. The experiments revealed that the N-terminal region is essential for proteolytic activity of the enzyme whereas deletions of the C-terminal regions had no significant effect on proteolytic activity. In addition, proteolytic activities of the enzymes were only detected after export into the culture supernatant. A biochemical characterization of the different active proteases was performed
Misplaced Women?
No full textMisplaced Women? is an ongoing interdisciplinary art project (2009-2017) by Tanja Ostojić that has been conceived as both an internet—platform and a real platform organized in public spaces in the cities across the globe to discuss the issues of migration, displacement, security, privacy, and exposure. It is manifested in a series of performances by the author herself, as well as delegated performances, individual or group performances predominantly by women, and performance workshops conducted by Tanja Ostojić herself. Essentially, the performance score might include unpacking, rummaging and detailed searching of the entire content, pockets, purses, wallets, personal suitcases and bags on sites that are relevant to migration, such as airports, train stations, Western Union Money Transfer services, police stations for foreigners who want to obtain residence permits, etc. Participants performing at authentic locations might repeat similar actions that build upon the basic proposal of the Misplaced Women? concept, i.e. they deal with positions andexperiences of people in transit, migration, and exile.Misplaced Women? is an ongoing interdisciplinary art project (2009-2017) by Tanja Ostojić that has been conceived as both an internet—platform and a real platform organized in public spaces in the cities across the globe to discuss the issues of migration, displacement, security, privacy, and exposure. It is manifested in a series of performances by the author herself, as well as delegated performances, individual or group performances predominantly by women, and performance workshops conducted by Tanja Ostojić herself. Essentially, the performance score might include unpacking, rummaging and detailed searching of the entire content, pockets, purses, wallets, personal suitcases and bags on sites that are relevant to migration, such as airports, train stations, Western Union Money Transfer services, police stations for foreigners who want to obtain residence permits, etc. Participants performing at authentic locations might repeat similar actions that build upon the basic proposal of the Misplaced Women? concept, i.e. they deal with positions andexperiences of people in transit, migration, and exile
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