Universität Stuttgart
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Item Open Access Precision 3D‐printed cell scaffolds mimicking native tissue composition and mechanics(2020) Erben, Amelie; Hörning, Marcel; Hartmann, Bastian; Becke, Tanja; Eisler, Stephan A.; Southan, Alexander; Cranz, Séverine; Hayden, Oliver; Kneidinger, Nikolaus; Königshoff, Melanie; Lindner, Michael; Tovar, Günter E. M.; Burgstaller, Gerald; Clausen‐Schaumann, Hauke; Sudhop, Stefanie; Heymann, MichaelCellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell‐ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell‐ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two‐photon stereolithography is adopted to print up to mm‐sized high‐precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein‐based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two‐pass printing or post‐print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7‐300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D‐lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single‐cell and tissue dynamics in response to defined mechanical and bio‐molecular cues and is ultimately scalable to full organs.Item Open Access Construction of robust Escherichia coli strains for large-scale production(2022) Ziegler, Martin; Takors, Ralf (Prof. Dr.-Ing.)The biotechnical production of many fine chemicals, proteins or pharmaceuticals depends on large-scale microbial cultivations. Due to limited mixing, heterogeneities in process relevant parameters such as nutrient concentrations arise in such fermentations. Escherichia coli (E. coli) is a model organism frequently used in the biotechnological industry. If E. coli is cultivated under heterogeneous conditions, biological reactions of the microorganism result in reduced process performance. Since large-scale fermentations are not economically feasible in academic settings, scale-down reactors that mimic aforementioned heterogeneities are used to investigate heterogenous fermentations. Previous studies in scale-down reactors unraveled that, depending on the process strategy, the unstable supply of a limiting primary carbon or nitrogen source such as glucose or ammonium is one of the underlying causes of process performance loss. Low concentrations of glucose or ammonium elicit the stringent response as a biological starvation reaction which comprises extensive transcriptional reactions. In the first project that contributes to this thesis, the regulatory and transcriptional reactions of the strains E. coli MG1655 and E. coli SR to repeated exposure to ammonium starvation zones were examined in a scale-down reactor. The scale-down reactor followed a two-compartment approach and consisted of a stirred tank reactor and a plug-flow reactor simulating passage through a starvation zone. E. coli SR is a strain with modulated stringent response. It was observed that short-term starvation stimuli do not trigger this regulatory program in E. coli SR and the transcriptional reaction was noticeably reduced. Long-term adaptation of the strain to repeated cycles of limitation and starvation also clearly differed from E. coli MG1655. Despite lack of the stringent response, E. coli SR showed no deficits in the assimilation of the limiting ammonium or in biomass yield on ammonium. In the second project of this thesis, a series of deletion strains with robust phenotype against glucose starvation zones were constructed. Candidate genes were identified and successively removed from the genome of E. coli MG1655 by Recombineering. The fundamental growth parameters of the strains were determined in shaking flask fermentations and no noticeable differences compared to E. coli MG1655 were found. Chemostat cultivations in a scale-down reactor with glucose as the limiting nutrient source revealed that the final strain of the deletion series, E. coli RM214, had a significantly lower maintenance coefficient under heterogeneous conditions than E. coli MG1655. Moreover, in an exemplary heterologous protein productionscenario E. coli RM214 rhaB- pJOE4056.2_tetA proved to be more robust to heterogeneities and showed a significantly higher product yield than E. coli MG1655 rhaB- pJOE4056.2_tetA. In the third project of this thesis, the production of pyruvate in E. coli MG1655 by inhibition of pyruvate dehydrogenase through CRISPR interference was investigated. A central goal was to achieve the stable production in nitrogen-limited conditions. For this, different target sequences in the operon pdhR-aceEF-lpd were tested and the strains cultivated in shaking flask fermentations. All tested target sequences were generally suitable to trigger the accumulation of pyruvate. Combined CRISPR interference against two target sequences did not lead to an increased pyruvate yield in most cases. In addition, the strains E. coli MG1655 pdCas9 psgRNA_aceE_234 and E. coli MG1655 pdCas9 psgRNA_aceE_234_pdhR_329 were characterized in two phase fermentations in lab-scale reactors. The initial phase was an unlimited exponential growth phase and was followed by an ammonium-limited production phase. E. coli MG1655 pdCas9 psgRNA_aceE_234 only produced pyruvate during the exponential phase, and reuptake of pyruvate occurred in the second phase. In contrast, E. coli MG1655 pdCas9 psgRNA_aceE_234_pdhR_329 stably produced pyruvate during the exponential and the ammonium-limited phase and is a potential chassis strain for the growth-decoupled production of pyruvate derived bioproducts. The overarching research issues of the projects were the characterization of strains in heterogeneous conditions and the development of new strategies to improve their performance. The collected data leads me to conclude that the construction of robust microbial strains for large-scale applications is both expedient and feasible. Tailored genetic modifications are the method of choice to achieve this goal. Furthermore, suitable genetic constructs offer promising possibilities for the stable growth-decoupled production of chemicals in nitrogen-limited conditions.Item Open Access Lokalisation, Speicherung und Synthese von Polyphosphat in Agrobacterium tumefaciens C58(2021) Hellenbroich, Celina; Jendrossek, Dieter (apl. Prof. Dr. rer. nat.)Polyphosphat (PolyP) besitzt eine ubiquitäre Verbreitung und erfüllt, je nach Organismus, unterschiedliche und extrem vielfältige Aufgaben. In Prokaryonten liegt PolyP in sogenannten Granula vor, während in einzelligen Eukaryonten, eine Membran das PolyP von dem Cytoplasma abtrennt. Vorangegangene Arbeiten weisen darauf hin, dass sogenannte Acidocalcisomen, eben jene membranumschlossene PolyP-Speicher aus Eukaryonten, auch in dem Bodenbakterium Agrobacterium tumefaciens vorhanden sein könnten. Die vorliegende Arbeit zeigt jedoch, dass sich in A. tumefaciens, wie in Bakterien übliche, PolyP-Granula befinden, die nicht von einer Membran umschlossen sind. Im weiteren Verlauf wurde die Synthese von PolyP sowie die Lokalisation der Polyphosphatkinasen (PPKs) und anderer aus der Literatur bekannter, PolyP-assoziierter Proteine untersucht. Die PPK1At stellte sich hierbei als PolyP-Syntheseenzym heraus. Es folgte eine biochemische Charakterisierung der PPKs in vitro, bei der für die PPK2At, neben der Bildung von NDP und NTP, eine oligophosphorylierende Funktion bis hin zu nonaphosphorylierten Nukleosiden entdeckt wurde. Außerdem stellte sich heraus, dass das PolyP-Granulum während des Zellzyklus wanderte und vielleicht durch die PPK1At mit der DNA assoziiert sein könnte. Aufgrund dieser Erkenntnisse konnte ein Modell des PolyP-Granulums und den in dieser Arbeit identifizierten, assoziierten Proteinen erstellt werden. Eine Deletion der ppk1 hatte zudem Auswirkungen auf die Zellmorphologie, die Infektionsrate von Pflanzenzellen und die Generationszeit von A. tumefaciens.Item Open Access Studien zur biotechnologischen Anwendung und ökologischen Funktion von Pyrrolochinolinchinon(PQQ)-abhängigen Alkoholdehydrogenasen(2021) Wehrmann, Matthias; Hauer, Bernhard (Prof. Dr.)Item Open Access Microbial biostabilization in fine sediments(2022) Gerbersdorf, Sabine Ulrike; Wieprecht, Silke (Prof. Dr.-Ing.)Microbial biostabilization has increasingly received attention over the last years due to its significance for the dynamics of fine sediments in fluvial and coastal systems with implications for ecology, economy and human-health. This habilitation thesis highlights the contributions of the applicant and her team to this multi-disciplinary research area and is based on eight core publications that are presented in seven chapters. First, the topic of biofilm and biostabilization is introduced and second, the materials and methods applied are presented before own research findings are discussed. To start with, the stabilization potential of heterotrophic bacterial assemblages has been emphasised as well as the adhesive properties of the protein moieties within the EPS (extracellular polymeric substances) that are more significant than previously thought. Furthermore, the engineering potential of estuarine prokaryotic and eukaryotic assemblages has been studied separately and combined to reveal the effective cooperation of mixed biofilm that resulted in highest substratum stabilization although the effects were not clearly synergistic (=more than additive). The significance of biostabilization could be evidenced as well for freshwaters where highest adhesive capacity and sediment stability occurred during spring. Microbial community composition differed accordingly to result in mechanically highly diverse biofilm. Moreover, the importance of two of the most influential abiotic conditions, light intensity and hydrodynamics, was shown for biofilm growth, species composition and functionality - here biostabilization. In order to test adhesive properties at the relevant mesoscale (mm-cm) but non-destructively and highly sensitive, MagPI (Magnetic Particle Induction) has been applied. The last chapter concerns technical aspects to further improve its performance while demonstrating the impact of material and geometry and the importance of both, magnetic field strength and field gradient for the physics of the MagPI approach.Item Open Access Resolving heterogeneities in single and multiphase bioreactor systems - Predictive modelling tools towards successful scale-up(2020) Kuschel, Maike; Takors, Ralf (Prof. Dr.-Ing.)Item Open Access Numerische Simulationen von bioinspirierten und natürlichen Materialien(2021) Schäfer, Immanuel; Schmauder, Siegfried (Prof. Dr. rer. nat. Dr. h. c.)Die Arbeit befasst sich mit Finite-Elemente-Simulationen und Modellierungen von natürlichen und bioinspirierten Materialien. Die bioinspirierten Materialien sind künstliche Materialien, die ein oder mehrere Prinzipien der biologischen Vorbilder erfolgreich umgesetzt haben. Die natürlichen Materialien sind biologischen Ursprungs. Zu Beginn benötigt man immer eine funktionelle Analyse des Systems, um dann die notwendigen Reduzierungen für die Modellierungen herstellen zu können. Wenn das Modell steht, werden Eigenschaften wie zum Beispiel die Dämpfung oder der Widerstand gegen eine lineare Krafteinwirkung in Finite-Elemente-Simulationen analysiert. Die daraus gewonnenen Ergebnisse werden zur Beurteilung des Versagenverhaltens (z. B. des natürlichen Materials) oder zur Erklärung der Eigenschaften des bioinspirierten Materials genutzt. Die vorliegende Arbeit ist in die Analyse und die Diskussion der Ergebnisse verschiedener Materialien in neun Veröffentlichungen unterteilt. Zu Beginn werden die Ergebnisse des ersten bionischen Projektes präsentiert sowie die Lichtaufnahme, Lichtleitung- und Lichtverarbeitungsmöglichkeiten einer sukkulenten Art, der Fensterpflanze diskutiert. Die Perlmuttschale einer Schnecke bzw. ihr bioinspiriertes Produkt ist das Thema der anschließend vorgestellten Arbeiten. Daraus hervor ging auch die Veröffentlichung zu Enzymen und deren Bindungsaffinitäten zu Zinkoxid. Die Schale der Pomelo, die die kiloschwere Frucht beim Herunterfallen von den ca. 15 m hohen Bäumen vor Beschädigungen schützt, ist das Vorbild für einen Metallschaum im nächsten Beispiel. Als letztes werden die Vorbilder Kokosnuss und Seeigelstachel präsentiert, die im Rahmen eines Transregios (einem von der Deutschen Forschungsgemeinschaft (DFG) geförderten Sonderforschungsbereich, TRR 141) betrachtet wurden. Für den TRR 141 wurde auch eine Ausstellung im Naturkundemuseum Stuttgart organisiert. Im Kapitel 12 wird eine Präsentationsstation beschrieben, die mit NFC(Near-Field-Communication)-Chips gesteuert werden kann. Die Präsentationsstation wurde im Rahmen dieser Arbeit entwickelt und mit dem Museum zusammen gebaut. Mit ihr werden Filme und Präsentationen zu dem Thema des TRR mit Ausstellungsgegenständen (z. B. eine 3D gedruckte Struktur des Seeigelstachels in Vergrößerung) kombiniert. Auf diese Weise zeigt die vorliegende kumulative Dissertation einen Überblick über die gesamten bionischen Arbeiten, die am Institut für Materialprüfung, Werkstoffkunde und Festigkeitslehre bei Herrn Prof. Dr. rer. nat. Dr. h. c. Siegfried Schmauder mit meiner Beteiligung entstanden sind.Item Open Access Eclectic characterisation of chemically modified cell-derived matrices obtained by metabolic glycoengineering and re-assessment of commonly used methods(2020) Keller, Silke; Liedek, Anke; Shendi, Dalia; Bach, Monika; Tovar, Günter E. M.; Kluger, Petra J.; Southan, AlexanderAzide-bearing cell-derived extracellular matrices (“clickECMs”) have emerged as a highly exciting new class of biomaterials. They conserve substantial characteristics of the natural extracellular matrix (ECM) and offer simultaneously small abiotic functional groups that enable bioorthogonal bioconjugation reactions. Despite their attractiveness, investigation of their biomolecular composition is very challenging due to the insoluble and highly complex nature of cell-derived matrices (CDMs). Yet, thorough qualitative and quantitative analysis of the overall material composition, organisation, localisation, and distribution of typical ECM-specific biomolecules is essential for consistent advancement of CDMs and the understanding of the prospective functions of the developed biomaterial. In this study, we evaluated frequently used methods for the analysis of complex CDMs. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and (immune)histochemical staining methods in combination with several microscopic techniques were found to be highly eligible. Commercially available colorimetric protein assays turned out to deliver inaccurate information on CDMs. In contrast, we determined the nitrogen content of CDMs by elementary analysis and converted it into total protein content using conversion factors which were calculated from matching amino acid compositions. The amount of insoluble collagens was assessed based on the hydroxyproline content. The Sircol™ assay was identified as a suitable method to quantify soluble collagens while the Blyscan™ assay was found to be well-suited for the quantification of sulphated glycosaminoglycans (sGAGs). Eventually, we propose a series of suitable methods to reliably characterise the biomolecular composition of fibroblast-derived clickECM.Item Open Access Entwicklung eines Verfahrens zur Herstellung von Propylenoxid und Phenol mittels Ganzzellbiokatalyse unter Nutzung der Methan-Monooxygenase des Organismus Methylosinus trichosporium OB3b(2022) Derwenskus, Ilka Madeleine; Hirth, Thomas (Prof. Dr.)Item Open Access Gold nanoparticle-mediated DNA origami nanoarchitectures(2024) Peil, Andreas; Na Liu, Laura (Prof. Dr.)Since its origin in the 1980s, DNA (deoxyribonucleic acid) nanotechnology has established itself as a captivating nanofabrication technique with ever increasing impact that combines aspects from physics, chemistry, and biology to construct artificial nanosystems by means of molecular self assembly. Within the field of DNA nanotechnology, the DNA origami technique represents one of the most versatile fabrication tools to craft functional two-dimensional (2D) and three-dimensional (3D) nanostructures from the bottom up. These structures offer precisely tailored geometries along with programmable functions, featuring positional addressability with sub-5 nm resolution and exceptional spatiotemporal accuracy. This thesis discusses strategies to employ the DNA origami technique to assemble intricate hybrid nanosystems with synergistically integrated gold nanoparticles (AuNPs). The AuNPs take over different roles; they grant (i) structural and (ii) functional features and enable the (iii) optical monitoring of the systems. This approach allows the fabrication of nanostructures piece by piece to explore their structural and functional properties at the nanoscale in detail. The first publication covers different strategies for the hierarchical assembly of topological DNA origami structures using a AuNP-templated self-assembly approach. The assembly of [2], [3], and [4]catenanes with interconnecting AuNPs is elucidated. The AuNPs can be controllably released to disconnect the individual rings, leaving only the mechanical bond of the catenane chain. In the second publication, a dynamic AuNP-DNA origami gear system is presented that is designed to emulate a planetary gearset with precise spatiotemporal control over its rotation dynamics. The AuNPs serve three crucial tasks. They (i) structurally link the origami ring modules, (ii) mediate the rotation and (iii) enable the real time optical tracking of the rotation via fluorescence spectroscopy. The system enables tightly orchestrated and programmable bidirectional rotations. In the third publication, reconfigurable chiral metastructures comprising multiple plasmonic particles that are accurately positioned in a helical manner around a DNA origami template are discussed. The implementation of a DNA ‘swingarm strategy’ enables the simultaneous and efficient relocation of multiple closely spaced AuNPs over large distances to precisely tune the chiroptical response of the system. The presented publications illustrate the beneficial synergies between DNA origami systems and rationally integrated AuNPs with the aim to advance and expand the application spectrum of these hybrid nanosystems within their scientific disciplines.