Monitoring and control of E. coli cell integrity.

Soluble expression of recombinant proteins in E. coli is often done by translocation of the product across the inner membrane (IM) into the periplasm, where it is retained by the outer membrane (OM). While the integrity of the IM is strongly coupled to viability and impurity release, a decrease in OM integrity (corresponding to increased "leakiness") leads to accumulation of product in the extracellular space, strongly impacting the downstream process. Whether leakiness is desired or not, differential monitoring and control of IM and OM integrity are necessary for an efficient E. coli bioprocess in compliance with the guidelines of Quality by Design and Process Analytical Technology. In this review, we give an overview of relevant monitoring tools, summarize the research on factors affecting E. coli membrane integrity and provide a brief discussion on how the available monitoring technology can be implemented in real-time control of E. coli cultivations

An assessment of heterosexuals\u27 perceived risk for HIV infection

Microalgae are a sustainable source of lipids. A commonly used strategy for lipid accumulation in microalgae is a two-step batch cultivation, with a growth phase followed by a nitrogen starvation phase. A problem with this process is the decrease in photosynthetic efficiency during the nitrogen starvation phase, which leads to low lipid productivities. In this research, a new process strategy was studied with the aim to improve lipid productivity of the microalgae Nannochloropsis gaditana. The nitrogen concentrations were chosen to assure consumption of most part of the nitrogen during the night. An improvement of the photosystem II maximum quantum yield and an increase in the dry weight and TAG concentration was achieved from day 7 of nitrogen starvation onwards when the culture was fed with nitrogen each night compared to a culture without nitrogen addition. Consequently, the time-average TAG yield on light was also higher after 7 days of nitrogen starvation. However, since the maximal time-averaged triacylglycerol (TAG) yield on light was reached after 3 days of nitrogen starvation, the improved photosynthetic activity did not lead to an increase of the maximal time-averaged TAG yield on light. The culture with nitrogen addition had a higher protein concentration (1.1 compared to 0.7 g L−1), showing that the added nitrogen was mainly used for protein production. A higher chlorophyll a content (2.0 compared to 0.8 μg mg−1) showed improved photosystem and that a small part of nitrogen was used for chlorophyll a. Small nightly nitrogen additions during batch cultivation of nitrogen starved N. gaditana did result in improvement in photosystem II maximal quantum yield, biomass concentration, TAG production and a higher time-averaged maximal TAG yield on light, after 7 days of nitrogen starvation.</p

Extracellular recombinant protein production with Escherichia coli

Kumulative Dissertation aus sechs ArtikelnEscherichia coli ist eines der gängigsten Expressionssysteme für rekombinante Proteine. Zu seinen Vorteilen zählen hohe Wachstumsraten, einfache genetische Manipulierung und geringes Kontaminationsrisiko. Ein signifikanter Nachteil ist jedoch, dass das Produkt intrazellulär akkumuliert. Während der Expression im Zytoplasma wird der Apparat für Proteinfaltung oftmals überladen, was zu physiologischem Stress und zur Bildung von unlöslichen Inclusion-Bodies führt. Obwohl durch unlösliche Expression hohe Produktivität und Reinheit erzielt werden können, bedarf die Aufreinigung von Inclusion-Bodies komplexer Solubilisierungs- und Rückfaltungsschritte. Um die Löslichkeit, Stabilität und Aktivität des Produkts zu verbessern, kann es durch die innere Membran in das Periplasma geschleust werden, indem eine entsprechende Signalsequenz angehängt wird. Translokation ist aufgrund der oxidativen Umgebung des Periplasmas besonders wichtig für die korrekte Faltung von Proteinen mit Disulfidbrücken. Dennoch werden periplasmatische Proteine durch die äußere Membran im Zellinneren gehalten und müssen durch Disruption der Zellhülle extrahiert werden. In aktuellen industriellen Verfahren geschieht dies durch komplette Zelllyse, was die Freisetzung von zytoplasmatischen sowie periplasmatischen Wirtsproteinen, DNA und Lipiden zur Folge hat. Diese Unreinheiten erschweren die folgenden Reinigungsschritte und führen somit zu einem Engpass im Downstream-Prozess. Diese Limitierung kann durch extrazelluläre Proteinproduktion überwunden werden. Außerdem kann diese den Übergang zu kontinuierlichen Prozessen mit E. coli erleichtern.Extrazelluläre Proteinproduktion kann zustande gebracht werden, indem die Durchlässigkeit der äußeren Membran selektiv erhöht wird und damit das Produkt in das Medium austritt (sog. „Leakiness“). Es gibt jedoch noch immer keine etablierten, industriell anwendbaren Methoden für selektive Extraktion periplasmatischer Proteine. Die Permeabilität der äußeren Membran kann allerdings auch im Upstream-Prozess erhöht werden. Durch Genmanipulation wurde bisher eine Reihe an Stämmen entwickelt, die permanente Leakiness aufweisen. Der Großteil dieser Systeme weist jedoch vermindertes Wachstum oder Viabilität auf und es gibt wenig Wissen darüber, wie Leakiness in solchen Stämmen kontrolliert werden kann. Ungeachtet der Methode zur Erhöhung der äußeren Membranpermeabilität kommt es oft zur gleichzeitigen Desintegrierung der inneren Membran, also zur Lyse. Daher werden Echtzeit-Methoden für differenziertes Monitoring der inneren und äußeren Membranpermeabilität benötigt, sowie Strategien um diese zu kontrollieren. Dies würde extrazelluläre Proteinproduktion im Sinne von Quality by Design ermöglichen.In dieser Dissertation wurden verschiedene Aspekte der extrazellulären Proteinproduktion mit E. coli untersucht.Die Literatur über vorhandene Methoden um die Zellintegrität von E. coli zu messen und zu kontrollieren wurde studiert und zusammengefasst. Während es kaum Berichte über Echtzeit-Monitoring von Leakiness und Lyse gibt, sind einige Technologien verfügbar, welche zu diesem Zweck eingesetzt werden könnten. Hinsichtlich der Kontrolle der Zellintegrität besteht die Hürde zur Umsetzung fortgeschrittener Methoden darin, dass mechanistisches Verständnis vom Einfluss der Prozessparameter auf die Zellhülle fehlt.Ein Pulsed-Electric-Fields-Verfahren wurde als neuartige Methode zur kontinuierlichen selektiven Extraktion von periplasmatischem Protein aus dem konventionellen Stamm E. coli BL21(DE3) untersucht. Es wurde gezeigt, dass dieser Prozess selektive Produktgewinnung bei geringer Lyse gewährleistet und damit eine vielversprechende Methode für industrielle Anwendungen ist.Weiters wurde der kürzlich entwickelte Stamm E. coli X-press auf seine Anwendbarkeit für extrazelluläre Proteinproduktion untersucht. Der Einfluss von Prozessparametern auf Wachstum, Produktivität, Leakiness und Lyse wurde analysiert. E. coli X-press zeigte hohe äußere Membranpermeabilität bei hoher Viabilität und stellt somit ein vielversprechendes System für extrazelluläre Produktionsverfahren dar. Zudem wurde die ökonomische und ökologische Auswirkung des X-press Stammes auf den frühen Downstream-Prozess untersucht und mit einem intrazellulären Prozess verglichen. Mittels Reinigungsexperimenten und Prozessmodellierung wurden Kosten und Ressourcenverbrauch geschätzt. Dadurch konnten die Vorteile der extrazellulären Produktion mit E. coli X-press für den Downstream-Prozess demonstriert werden.Zuletzt wurde die Anwendbarkeit zweier Methoden für Monitoring der Zellintegrität von E. coli untersucht. Einerseits wurde gezeigt, dass das Einsetzen von Leakiness mittels hochauflösender Dichtemessungen des Kulturmediums detektiert werden kann und diese Methode eine wertvolle Ergänzung in der Analysen-Toolbox ist. Zusätzlich wurde Attenuated-Total-Reflectance-Fourier-Transform-Infrarot-Spektroskopie in Kombination mit chemometrischen Analysen als Monitoring-Methode getestet. Deren Potential sowie Vorbehalte für den Einsatz in E. coli-Kulturen wurden aufgezeigt.Das Wissen und die Methoden, die im Rahmen dieser Dissertation generiert wurden, tragen zu Verständnis und Kontrolle der Zellintegrität von E. coli bei und werden zukünftige Bioprozesse mit diesem Mikroorganismus erleichtern.Escherichia coli is among the most popular expression hosts for recombinant proteins as it offers several advantages, such as high growth rates, simple genetic manipulation and low risk of contamination. However, a major drawback is that the product accumulates intracellularly. During expression in the cytoplasm, the folding machinery is often overloaded, leading to physiological stress and the formation of insoluble inclusion bodies. Although high productivity and purity can be achieved by insoluble expression strategies, downstream processing of inclusion bodies entails complex solubilization and refolding procedures. To improve protein solubility, stability and activity, the product may be translocated through the inner membrane into the periplasm by adding an appropriate signal sequence. Due to the oxidative environment of the periplasm, translocation is particularly important for the correct folding of proteins containing disulfide bonds. Yet, periplasmic proteins are usually retained by the outer membrane and must be extracted by disrupting the cell envelope. In current industrial scenarios, this is done by complete cell lysis, which leads to the release of host cell protein (both periplasmic and cytoplasmic), DNA and membrane lipids. These impurities severely hamper subsequent purification steps and thus create a bottleneck in the downstream process. Extracellular protein production can overcome this limitation. Furthermore, it may facilitate the transition to continuous bioprocessing with E. coli.Extracellular protein production can be achieved by selective permeabilization of the outer membrane and consequent leakage of the product to the medium. However, there is still a lack of industrially applicable methods for selective extraction of periplasmic proteins. Outer membrane permeability may also be enhanced in the upstream process. Genetic engineering of the E. coli cell envelope has been employed for the construction of various mutants displaying permanent leakiness. However, most of these hosts display impaired growth or viability and there is limited knowledge on how to control leakiness during the cultivation of such strains. Regardless of the method for outer membrane permeabilization, disintegration of the inner membrane (i.e. lysis) often co-occurs. Thus, methods for rapid, differential monitoring of inner and outer membrane integrity are needed, as well as strategies to control leakiness. This would allow extracellular production scenarios aligned with the principles of Quality by Design. In this Thesis, several aspects of extracellular protein production with E. coli were investigated.The literature on available methods for monitoring and controlling E. coli cell integrity during the upstream process was studied and reviewed. While reports about real-time monitoring of leakiness and lysis are scarce, a variety of technologies exist that might be suitable for this purpose. Regarding the control of E. coli cell integrity, the implementation of advanced methods is still hindered by limited mechanistic understanding of the effect of process parameters on the cell envelope.Pulsed electric fields treatment was assessed as a novel method for continuous selective extraction of periplasmic protein from the conventional, “non-leaky” strain E. coli BL21(DE3). It was shown that this procedure yields selective product recovery while keeping lysis low. Thus, this method is promising for industrial applications.Moreover, the utility of the recently developed E. coli X-press strain for extracellular protein production was examined. The influence of process parameters on the host’s growth, productivity, leakiness and lysis was studied. E. coli X-press displayed high outer membrane permeability while maintaining viability, constituting a promising expression system for extracellular production scenarios. Furthermore, the ecological and economic impact of utilizing E. coli X-press on the early downstream process was analyzed and compared to an intracellular production process. Purification experiments were combined with process modeling to estimate costs and resource consumption. Thereby, the benefits of extracellular production with E. coli X-press for the downstream process were demonstrated.Finally, two methods were studied for their applicability to monitor E. coli cell integrity. On one hand, it was shown that high-resolution measurements of culture medium density can be used to detect the onset of leakiness presenting a valuable addition to the ensemble of monitoring techniques. In addition, in-line Attenuated-Total-Reflectance Fourier-Transform-Infrared spectroscopy in combination with chemometrics was assessed as a monitoring tool. The potential of this method as well as its limitations for use in E. coli cultivations were demonstrated.Overall, the knowledge and methods generated in this Thesis contribute to the understanding and control of E. coli cell leakiness and will aid future bioprocessing with this microorganism.12

Scientific cultures of non-knowledge in the controversy over genetically modified organisms (GMO): the cases of molecular biology and ecology

The limits of scientific knowledge are an emerging problem in the debates about technological risk. In an exemplary analysis of the controversy surrounding genetically modified organisms (GMO), we show that the epistemic settings of two involved scientific disciplines – molecular biology and ecology – entail different types of non-knowledge and deal with non-knowledge differently. Both of these "scientific cultures of non-knowledge" are analysed along five criteria: the way of dealing with unforeseen events, the way of dealing with complexity and uncertainty, the temporal and spatial scales of knowledge, the de-and re-contextualisation of knowledge, and the epistemic (self-)reflexivity. The scientific culture of non-knowledge in molecular biology can be described as control-oriented, while that of ecology can be described as uncertainty-oriented. This difference is mirrored in the societal discourses and regulations concerning GMO. A greater variety of cultures of non-knowledge seems likely, which calls for further analysis.</jats:p

Scientific Nonknowledge and Its Political Dynamics: The Cases of Agri-Biotechnology and Mobile Phoning

While in the beginning of the environmental debate, conflicts over environmental and technological issues had primarily been understood in terms of "risk", over the past two decades the relevance of ignorance, or nonknowledge, was emphasized. Referring to this shift of attention to nonknowledge the article presents two main findings: first, that in debates on what is not known and how to appraise it different and partly conflicting epistemic cultures of nonknowledge can be discerned and, second, that drawing attention to nonknowledge in technology conflicts results in significant institutional effects and new constellations of actors in public debates. To illustrate and substantiate this political dynamics of nonknowledge we draw upon examples from the areas of agri-biotechnology and mobile phoning. In a first step, we develop in greater detail the concept of scientific cultures of nonknowledge and identify three such cultures involved in the social conflicts within the two areas. Subsequently, we analyze the specific dynamics of the politicisation of nonknowledge looking at the variety of actors involved and the pluralisation of perceptions and evaluations of what is not known. Then, we point out some of the institutional reactions to the political and cultural dynamics of scientific nonknowledge. We argue that the equal recognition of the diverse cultures of nonknowledge is a key prerequisite for socially legitimate and "robust" decision-making under conditions of politicised scientific nonknowledge. © The Author(s) 2010