Engineering Signal Transduction Pathways in Bacteria

Veränderte Proteine sind ein wertvolles Handwerkzeug, sowohl in der Grundlagenforschung als auch in der Industrie. Ein nächster Schritt wäre Veränderungen in biologischen Stoffwechselwegen, z.B. in der Signalweiterleitung, vorzunehmen. Dieses könnte die Grundlage für viele Anwendungen sein, z.B. in der Gentherapie um neue Eigenschaften in Organismen einzuführen. Viele biologische Prozesse sind einem hohen Rauschpegel unterworfen, was für viele Anwendungen nicht geeignet ist. So können z.B in einer Population von Zellen die Gene unterschiedlich stark aktiviert werden, was zu niedriger oder hoher Expression führen kann. Daher ist es wichtig beim Entwurf von neuen Signalwegen, dass der Output gut kontrolliert werden kann. Da man solch einer Herausforderung nicht einfach gerecht werden kann, ist es besser mit einfachen und gut analysierten Signalwegen, wie sie z.B. in E.coli vorkommen, anzufangen. Die Signalweiterleitung in E.coli erfolgt hauptsächlich in Zwei-Komponenten Systemen. Diese bestehen aus einer Kinase als Sensor und einem Regulatorprotein, welches normalerweise ein Transkriptionsfaktor ist, mit einigen Ausnahmen, wie z.B CheY aus dem Chemotaxis Signalweg. In dieser Arbeit wurden zwei gut charakterisierte Signalwege durch einen chimärischen Sensor verbunden, um so einen neuen Signalweg herzustellen: Die Ligandenbindungsdomäne des Aspartat Receptors, Tar, aus dem chemotaxischen Signalweg wurde mit der katalytischen Domäne des osmosensorischen EnvZ kombiniert, um den chimärischen Rezeptor Taz herzustellen. Taz kann die Genexpresion aktivieren durch Phosphorylierung des Antwort-Regulators OmpR, über den Porin Promotor, nach Bindung eines geeigneten Liganden, wie z.B. Aspartat. Ziel dieser Studie war es dieses System zu nutzen um einen Signalweg herzustellen, dessen Output durch einen Gen-Schaltkreis kontrolliert ist, und das Eingangssignal durch gezieltes Design zu ändern. Als Output wurde ein etabliertes GFP verwendet und unterschiedliche Schaltkreise wurden verwendet: a) Kompetition von OmpR-P mit dem TetR Repressor, expremiert von einem synthetischen Promotor, zur Aktivierung des pompC Promotors, b) Expression von Antisense RNA für das Reportergen (GFP) and c) einen bistabilen Schalter durch TetR und einem temperatur-abhängigen Protein CI, welches durch OmpR-P aktiviert wird und die größte Regulation des Outputs ist. Trotz dieser unterschiedlichen Ansätze war es nicht möglich ein stabiles Konstrukt zu erhalten mit ausreichender Promotor Stärke, um einen Effekt aufzuweisen im Vergleich zu dem einfachen Reporter pompC-GPP. Das Liganden-Design erfolgte mit Hilfe der Algorithmen PERLA und FoldX sowie dem Programm SwissPdbViewer, basierend auf der Kristallstruktur der periplasmatischen Domäne von Tar im Komplex mit Glutamat. Obwohl diese Methode sehr vielversprechend aussah, als sie für einige Mutaten in dem Wildtyp Chemotaxis System durchgeführt wurde, war sie nicht erfolgreich um die Liganden des Chimären Rezeptors zu ändern. Ein Grund hierfür könnte sein, dass der chimäre Rezeptor in der periplasmatischen Domäne eine andere Konformation als der Wildtype Rezeptor hat, als Folge der Fusion mit der cytoplasmatischen Domäne von EnvZ. Dies wird durch experimenteller Ergebnisse unterstützt, die zeigen, dass Tar und Taz nicht dieselben Erkennungs-Eigenschaften haben, so z.B. können gleiche Signale für Tar entgegengesetzte Antworten durch Taz hervorrufen. Dennoch, in dieser Studie wurden in großem Detail die Eigenschaften von Taz untersucht, was zur ersten Entdeckung von Liganden (Aminosäuren) führte, die den Rezeptor inhibieren können. Weiterhin konnte gezeigt werden, dass diese Inhibierung stereo-spezifisch ist und extrem stark ist, sogar stärker als dieBindung von Liganden wie Aspartat. Weiterhin ergaben die intrinsischen Eigenschaften des Taz-OmpR Systems, die in dieser Studie gefunden wurden, eine neue Ansicht für das Wildtyp-System: Die Aktivierung des EnvZ-OmpR Systems in E. coli während des Zellwachstums scheint über EnvZ zu erfolgen, und die katalytische Domäne von EnvZ ist nicht ausreichend um dafür. Der wichtigste Befund aber für das chemotaxische System ist, dass einige der inhibitorischen Aminosäuren für Taz zu einer neuen, komplizierten Antwort führten und somit die Existenz eines bisher uncharakterisierten Adaptation Signalweges aufgedeckt wude

Pattern-driven security, privacy, dependability and interoperability management of iot environments

Achieving Security, Privacy, Dependability and Interoperability (SPDI) is of paramount importance for the ubiquitous deployment and impact maximization of Internet of Things (IoT) applications. Nevertheless, said requirements are not only difficult to achieve at system initialization, but also hard to prove and maintain at run-time. This paper highlights an approach to tackling the above challenges, through the definition of pattern language and a framework that can guarantee SPDI in IoT orchestrations. By integrating pattern reasoning engines at the various layers of the IoT infrastructure, and a machine-processable representation of said pattern through Drools rules, the proposed framework can provide ways to fulfill SPDI requirements at design time, and also provide the means to guarantee those SPDI properties and manage the orchestrations accordingly. Moreover, an application example of the framework is presented in an Industrial IoT monitoring environment

Noise in transcription negative feedback loops: simulation and experimental analysis

Negative feedback loops have been invoked as a way to control and decrease transcriptional noise. Here, we have built three circuits to test the effect of negative feedback loops on transcriptional noise of an autoregulated gene encoding a transcription factor (TF) and a downstream gene (DG), regulated by this TF. Experimental analysis shows that self-repression decreases noise compared to expression from a non-regulated promoter. Interestingly enough, we find that noise minimization by negative feedback loop is optimal within a range of repression strength. Repression values outside this range result in noise increase producing a U-shaped behaviour. This behaviour is the result of external noise probably arising from plasmid fluctuations as shown by simulation of the network. Regarding the target gene of a self-repressed TF (sTF), we find a strong decrease of noise when repression by the sTF is strong and a higher degree of noise anti-correlation between sTF and its target. Simulations of the circuits indicate that the main source of noise in these circuits could come from plasmid variation and therefore that negative feedback loops play an important role in suppressing both external and internal noise. An important observation is that DG expression without negative feedback exhibits bimodality at intermediate TF repression values. This bimodal behaviour seems to be the result of external noise as it can only be found in those simulations that include plasmid variation

Engineering prokaryotic gene circuits

Engineering of synthetic gene circuits is a rapidly growing discipline, currently dominated by prokaryotic transcription networks, which can be easily rearranged or rewired to give different output behaviours. In this review, we examine both a rational and a combinatorial design of such networks and discuss progress on using in vitro evolution techniques to obtain functional systems. Moving beyond pure transcription networks, more and more networks are being implemented at the level of RNA, taking advantage of mechanisms of translational control and aptamer–small molecule complex formation. Unlike gene expression systems, metabolic components are generally not as interconnectable in any combination, and so engineering of metabolic circuits is a particularly challenging field. Nonetheless, metabolic engineering has immense potential to provide useful biosynthesis tools for biotechnology applications. Finally, although prokaryotes are mostly studied as single cell systems, cell–cell communication networks are now being developed that result in spatial pattern formation in multicellular prokaryote colonies. This represents a crossover with multicellular organisms, showing that prokaryotic systems have the potential to tackle questions traditionally associated with developmental biology. Overall, the current advances in synthetic gene synthesis, ultra-high-throughput DNA sequencing and computation are synergizing to drive synthetic gene network design at an unprecedented pace

CIRCE: Architectural Patterns for Circular and Trustworthy By-Design IoT Orchestrations

The adoption of Internet of Things (IoT) devices, applications and services gradually transform our everyday lives. In parallel, the transition from linear to circular economic (CE) models provide an even more fertile ground for novel types of services, and the update and enrichment of legacy ones. To fully realize the potential of the interplay between IoT and CE, the design-time definition of IoT orchestrations with proven circularity properties, and the run-time management of these orchestrations based on said properties, is of paramount importance. Nevertheless, the circularity requirements and associated properties are not only difficult to achieve at the IoT orchestration design and deployment initialization phases, but also hard to prove and maintain at run-time. Motivated by this, this paper presents the CIRCE framework for circular and trustworthy by-design IoT orchestrations. The CIRCE approach leverages concepts from pattern-driven engineering, whereby patterns are used to encode proven dependencies between the Location, Condition, and Availability (LCA) properties of individual smart objects and corresponding properties of orchestrations (compositions) involving them. These are augmented by patterns encoding trustworthiness-related properties, namely Connectivity, Security, Privacy, Dependability, and Interoperability (CSPDI). Thereby, these patterns are used to generate IoT orchestrations with proven LCA and CSPDI properties, as needed, at design time. At runtime, these properties are monitored in real-time, leveraging reasoning engines deployed across system layers, triggering adaptations to return the deployed orchestration to the desired LCA and CSPDI states, when required. Details are provided on the above novel combination of IoT, CE and pattern-based engineering, along with a proposed architecture and implementation approach. Furthermore, an assessment of a proof-of-concept implementation is provided, validating the feasibility of the proposed approach

Safety-Security Co-Engineering Framework

Executive Summary:The advantages of a model-based approach for safety have been clear for many years now. However, security analysis is typically less formal and more ad-hoc; it may involve systematic processes but these are not generally tied into a formal model-based development and analysis process in the same way that safety can be.Task 4.3 of the SESAME project, Safety/Security Co-Engineering, sets out to remedy this by investigating a holistic co-engineering approach that integrates both of these different concerns.In this report we therefore present a combined safety/security co-engineering framework based on the ODE, the metamodel that serves as a basis for the EDDI dependability management concept. The ODE acts as a common ontology for both safety and security, establishing equivalencies between key concepts and allowing joint analyses to take place in which failures can be incorporated into security analysis and attacks into safety analysis. The combined results indicate the causes and consequences of hazardous events regardless of whether they originate from safety or security issues, and the same risk estimation applies to them all. While developed for design-time usage, this framework paves the way for the generation of combined safety/security artefacts at runtime as well. The common approach means that specification of requirements, event monitors, diagnostic engines, and responses/actions can take advantage of both safety and security information stored in the design-time models.To demonstrate the approach, we apply it to a drone-based case study derived from two of the SESAME use cases

Low thrust propulsion in a coplanar circular restricted four body problem

This paper formulates a circular restricted four body problem (CRFBP), where the three primaries are set in the stable Lagrangian equilateral triangle configuration and the fourth body is massless. The analysis of this autonomous coplanar CRFBP is undertaken, which identies eight natural equilibria; four of which are close to the smaller body, two stable and two unstable, when considering the primaries to be the Sun and two smaller bodies of the solar system. Following this, the model incorporates `near term' low-thrust propulsion capabilities to generate surfaces of articial equilibrium points close to the smaller primary, both in and out of the plane containing the celestial bodies. A stability analysis of these points is carried out and a stable subset of them is identied. Throughout the analysis the Sun-Jupiter-Asteroid-Spacecraft system is used, for conceivable masses of a hypothetical asteroid set at the libration point L4. It is shown that eight bounded orbits exist, which can be maintained with a constant thrust less than 1:5 10􀀀4N for a 1000kg spacecraft. This illustrates that, by exploiting low-thrust technologies, it would be possible to maintain an observation point more than 66% closer to the asteroid than that of a stable natural equilibrium point. The analysis then focusses on a major Jupiter Trojan: the 624-Hektor asteroid. The thrust required to enable close asteroid observation is determined in the simplied CRFBP model. Finally, a numerical simulation of the real Sun-Jupiter-624 Hektor-Spacecraft is undertaken, which tests the validity of the stability analysis of the simplied model

Maritime route of colonization of Europe

The Neolithic populations, which colonized Europe approximately 9,000 y ago, presumably migrated from Near East to Anatolia and from there to Central Europe through Thrace and the Balkans. An alternative route would have been island hopping across the Southern European coast. To test this hypothesis, we analyzed genome-wide DNA polymorphisms on populations bordering the Mediterranean coast and from Anatolia and mainland Europe. We observe a striking structure correlating genes with geography around the Mediterranean Sea with characteristic east to west clines of gene flow. Using population network analysis, we also find that the gene flow from Anatolia to Europe was through Dodecanese, Crete, and the Southern European coast, compatible with the hypothesis that a maritime coastal route was mainly used for the migration of Neolithic farmers to Europe