Facile promoter deletion in Escherichia coli in response to leaky expression of very robust and benign proteins from common expression vectors

BACKGROUND: Overexpression of proteins in Escherichia coli is considered routine today, at least when the protein is soluble and not otherwise toxic for the host. We report here that the massive overproduction of even such "benign" proteins can cause surprisingly efficient promoter deletions in the expression plasmid, leading to the growth of only non-producers, when expression is not well repressed in the newly transformed bacterial cell. Because deletion is so facile, it might impact on high-throughput protein production, e.g. for structural genomics, where not every expression parameter will be monitored. RESULTS: We studied the high-level expression of several robust non-toxic proteins using a T5 promoter under lac operator control. Full induction leads to no significant growth retardation. We compared expression from almost identical plasmids with or without the lacI gene together in strains expressing different levels of LacI. Any combination without net overexpression of LacI led to an efficient promoter deletion in the plasmid, although the number of growing colonies and even the plasmid size - all antibiotic-resistant non-producers - was almost normal, and thus the problem not immediately recognizable. However, by assuring sufficient repression during the initial establishment phase of the plasmid, deletion was completely prevented. CONCLUSION: The deletions in the insufficiently repressed system are caused entirely by the burden of high-level translation. Since the E. coli Dps protein, known to protect DNA against stress in the stationary phase, is accumulated in the deletion mutants, the mutation may have taken place during a transient stationary phase. The cause of the deletion is thus distinct from the well known interference of high-level transcription with plasmid replication. The deletion can be entirely prevented by overexpressing LacI, a useful precaution even without any signs of stress caused by the protein

Fuel Dependent Heat Release Differences between Euro Diesel Fuel and RME in a HSDI Diesel Engine

In the degree that costs and demand of crude oil rise, diminish the economical disadvantages for alternative Diesel fuels, resulting in a variety of feasible substitutes. Diesel fuel substitutes have deviating exhaust emissions from conventional fuel. The methyl ester of rapeseed oil (known as RME/Biodiesel) is receiving increasing attention as an alternative fuel for Diesel engines. RME is a non-toxic, biodegradable, and renewable fuel with the potential to reduce engine exhaust emissions [1]. The main disadvantage for RME is its vaporisation and self ignition characteristics at low load conditions. Engine experiments were carried out at 4 bar IMEP with Euro Diesel fuel (EDF) as reference and RME. During these engine experiments EGR and injection pressure were varied. As a result, differences in exhaust emissions due to EGR, injection pressure and fuel type were observed. The objective of this work was to find answers for fuel dependent differences in indicated load and exhaust gas emissions. As combustion and emission formation of RME has not been fundamentally explained yet [2], a detailed analysis approach based on explanation models for fuel characteristics was chosen to explain the observed differences

Multimedia - was steckt hinter den Schlagworten

Die Schlagworte aus der Multimedia-Welt kennt jeder: Elektronische Post, Daten-Highway und virtuelle Unternehmen sind nur einige davon. Welche konkreten Auswirkungen hat die schöne neue Multimedia-Welt auf unseren Alltag? Was bringen die Anwendungen wirklich? Die rund 90 Teilnehmer einer Tagung Anfang Juni in Stuttgart wollten es genau wissen: Drei Tage lang informierten sie sich aus erster Hand über das Thema Multimedia. Für ihre diesjährige Fachtagung hatten die Veranstalter - Oregon Alumni Association e.V. und Fulbright Alumni e.V. - wieder namhafte Referenten gewinnen können. Mit den Fachtagungen unterstreichen die beiden Vereine, in denen sich ehemalige Teilnehmer des deutsch-amerikanischen Studentenaustausches zusammengeschlossen haben, ihren Anspruch, das Netzwerk der Ehemaligen zu stützen und die Grundideen des deutsch-amerikanischen Austausches zu pflegen

Detailed Heat Release Analyses With Regard To Combustion of RME and Oxygenated Fuels in an HSDI Diesel Engine

Experiments on a modern DI Diesel engine were carried out: The engine was fuelled with standard Diesel fuel, RME and a mixture of 85% standard Diesel fuel, 5% RME and 10% higher alcohols under low load conditions (4 bar IMEP). During these experiments, different external EGR levels were applied while the injection timing was chosen in a way to keep the location of 50% heat release constant. Emission analysis results were in accordance with widely known correlations: Increasing EGR rates lowered NOx emissions. This is explained by a decrease of global air-fuel ratio entailing longer ignition delay. Local gas-fuel ratio increases during ignition delay and local combustion temperature is lowered. Exhaust gas analysis indicated further a strong increase of CO, PM and unburned HC emissions at high EGR levels. This resulted in lower combustion efficiency. PM emissions however, decreased above 50% EGR which was also in accordance with previously reported results. Besides those similar trends, fuel dependent differences in indicated thermal efficiency as well as CO, HC, NOx and especially PM emissions were observed. These differences were evaluated by detailed heat release analysis and explanation models based upon fuel characteristics as fuel viscosity and fuel distillation curve. Fuel spray evaporation and heat release were influenced by these fuel characteristics. Due to these characteristics it was concluded that RME has a higher tendency to form fuel rich zones at low load conditions than the other tested fuel types. Moreover it was found that improved fuel spray vaporisation is an option to improve exhaust emissions at low load conditions

Floristic inventories and distribution maps of vascular plants in Germany from the period between 1945 and 2005

In den letzten sechs Jahrzehnten wurden in Deutschland rund 200 nationale, regionale und lokale Florenwerke publiziert. Die vorliegende Arbeit gibt einen Überblick über die in diesen Publikationen enthaltenen Daten und stellt eine Karte mit den Bearbeitungsgebieten regionaler Florenwerke vor. Ein bibliografischer Teil zeigt die wichtigsten Daten für jede Publikation. Oft durch qualifizierte Amateurbotaniker erarbeitet, bilden solche Florenwerke eine wichtige Datenbasis insbesondere für Programme zum Artenschutz sowie für die Erstellung von Roten Listen.Within the last six decades about 200 national, regional, and local floristic inventories, often including distribution maps, for vascular plants in Germany have been published. An overview about the data provided by these publications is given together with a map displaying the coverage of regional and local inventories. A bibliography lists the key data for each publication. Often compiled by skilled volunteer botanists, such floristic inventories constitute an essential database for the compilation of Red Data Books and for species conservation

MBMS—IP Multicast/Broadcast in 3G Networks

In this article, the Multimedia Broadcast and Multicast Service (MBMS) as standardized in 3GPP is presented. With MBMS, multicast and broadcast capabilities are introduced into cellular networks. After an introduction into MBMS technology, MBMS radio bearer realizations are presented. Different MBMS bearer services like broadcast mode, enhanced broadcast mode and multicast mode are discussed. Streaming and download services over MBMS are presented and supported media codecs are listed. Service layer components as defined in Open Mobile Alliance (OMA) are introduced. For a Mobile TV use case capacity improvements achieved by MBMS are shown. Finally, evolution of MBMS as part of 3GPP standardization is presented

New Aspects in the Differential Diagnosis and Therapy of Bladder Pain Syndrome/Interstitial Cystitis

Diagnosis of bladder pain syndrome/interstitial cystitis (BPS/IC) is presently based on mainly clinical symptoms. BPS/IC can be considered as a worst-case scenario of bladder overactivity of unknown origin, including bladder pain. Usually, patients are partially or completely resistant to anticholinergic therapy, and therapeutical options are especially restricted in case of BPS/IC. Therefore, early detection of patients prone to develop BPS/IC symptoms is essential for successful therapy. We propose extended diagnostics including molecular markers. Differential diagnosis should be based on three diagnostical “columns”: (i) clinical diagnostics, (ii) histopathology, and (iii) molecular diagnostics. Analysis of molecular alterations of receptor expression in detrusor smooth muscle cells and urothelial integrity is necessary to develop patient-tailored therapeutical concepts. Although more research is needed to elucidate the pathomechanisms involved, extended BPS/IC diagnostics could already be integrated into routine patient care, allowing evidence-based pharmacotherapy of patients with idiopathic bladder overactivity and BPS/IC

Innovative Bauteilgestaltung mit inneren Strukturen

Die neuen Fertigungsmöglichkeiten durch die Additive Fertigung ermöglicht es nicht nur topologisch neuartige Bauteile herzustellen, sondern auch Bauteile mit inneren Strukturen zu versehen, die der Bauteilbelastung angepasst sind oder anderen Funktionen Freiräume bieten. Ein Ansatz ist es durchlässige innere Strukturen, z. B. Gitterstrukturen (auch als Lattice Strukturen bezeichnet) einzusetzen und durch die damit geschaffenen großen inneren Flächen eine effiziente Bauteilkühlung zu realisieren. Anhand eines einfachen Beispiels wird durch Simulation und Experiment die Wirkung einer solchen Kühlung gezeigt. Als weiteres Anwendungsbeispiel wird der Einsatz verschiedener innere Strukturen zur festigkeitsgerechten Gestaltung gewichtsoptimierter Bauteile vorgestellt. In beiden Fällen wird die Gestaltung mit Hilfe von FE-Modellen experimentell begleitet.The new manufacturing possibilities offered by additive manufacturing not only allows to produce topologically novel components, but also enables to provide components with internal structures that are adapted to the component load or offer new possibilities for other functions. One approach is to use permeable internal structures, e. g. lattice structures, to realize efficient component cooling through the large internal surfaces created thereby. The effect of such a cooling is demonstrated by simulations and experiments using a simple example. As a further application example, the use of various internal structures for the strength-oriented design of weight-optimized components will be presented. In both cases the design is experimentally accompanied by FE models

Novel approach of high cell density recombinant bioprocess development: Optimisation and scale-up from microlitre to pilot scales while maintaining the fed-batch cultivation mode of E. coli cultures

<p>Abstract</p> <p>Background</p> <p>Bioprocess development of recombinant proteins is time consuming and laborious as many factors influence the accumulation of the product in the soluble and active form. Currently, in most cases the developmental line is characterised by a screening stage which is performed under batch conditions followed by the development of the fed-batch process. Performing the screening already under fed-batch conditions would limit the amount of work and guarantee that the selected favoured conditions also work in the production scale.</p> <p>Results</p> <p>Here, for the first time, high throughput multifactorial screening of a cloning library is combined with the fed-batch technique in 96-well plates, and a strategy is directly derived for scaling to bioreactor scale. At the example of a difficult to express protein, an RNase inhibitor, it is demonstrated that screening of various vector constructs and growth conditions can be performed in a coherent line by (i) applying a vector library with promoters and ribosome binding sites of different strength and various fusion partners together with (ii) an early stage use of the fed-batch technology. It is shown that the EnBase^®technology provides an easy solution for controlled cultivation conditions in the microwell scale. Additionally the high cell densities obtained provide material for various analyses from the small culture volumes. Crucial factors for a high yield of the target protein in the actual case were (i) the fusion partner, (ii) the use of of a mineral salt medium together with the fed-batch technique, and (iii) the preinduction growth rate. Finally, it is shown that the favorable conditions selected in the microwell plate and shake flask scales also work in the bioreactor.</p> <p>Conclusions</p> <p>Cultivation media and culture conditions have a major impact on the success of a screening procedure. Therefore the application of controlled cultivation conditions is pivotal. The consequent use of fed-batch conditons from the first screening phase not only shortens the developmental line by guarantying that the selected conditions are relevant for the scale up, but in our case also standard batch cultures failed to select the right clone or conditions at all.</p