Dokument: Development and implementation of (opto-) genetic tools in different eukaryotic organisms and application of synthetic biology approaches for studying plant signaling pathways
| Titel: | Development and implementation of (opto-) genetic tools in different eukaryotic organisms and application of synthetic biology approaches for studying plant signaling pathways | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=54980 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20211221-092356-8 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Hüsemann, Lisa Cristin [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Zurbriggen, Matias [Gutachter] Prof. Dr. Feldbrügge, Michael [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | The field of synthetic biology offers a completely new approach to unravel complex cellular mechanisms as well as to develop urgently required biotechnological processes. For this purpose, engineering principles are applied to design and implement molecular tools for the assembly of complex synthetic switches and networks. Over the years, many of these tools and switches have been established in mammalian cell systems, plants, bacteria and yeast. The basidiomycete fungus Ustilago maydis was not among these organisms, even though it is of great interest for studying fundamental cellular principles, like long-distance transport, and for a broad spectrum of biotechnological applications.
To fill this gap, we implemented a synthetic biology toolbox specifically designed to be applied in this microorganism. Therefore, we tested and designed tools for building multicistronic vectors, enabling the expression of more than one gene under control of a single promotor. Furthermore, we established different reporter genes and their corresponding assays to quantitatively analyze the functionality of all tools and systems, designed and constructed in the course of this work. These reporter genes can be applied for a multitude of current and future research attempts in this fungus. Having these basic tools at hand, we designed, built, implemented and characterized two chemically and three light-controllable gene expression systems in U. maydis. The tetracycline-regulated Tet-off system that was established here, showed an unexpected reverse mode of function, converting the off-system to an on-system. This system showed low basal activity levels and a high induction fold, making it a promising tool for regulating the expression of genes in this organism. Furthermore, we established a gene expression system which is negatively controllable in the presence of macrolide antibiotics. This system depends on the erythromycin-regulated binding of the E-protein from Escherichia coli to its operator sequence (ETR). Here we demonstrate a high level of controllability of gene expression in U. maydis, considering a low basal activity and a high reduction fold, provided by this system. The utilization of light as a regulator of cellular events in synthetic biology approaches is one of the most outstanding accomplishments of this field. Systems that are designed to integrate light signal inputs and generate an increased or reduced level of gene expression as an output, have been established in many eukaryotic organisms. In contrast to antibiotics or other drugs, the application of light as a regulating factor is offering a higher spatial and temporal control, causing less disadvantageous side effects. Here we report on the novel establishment of three blue light-controllable gene expression systems in U. maydis, and demonstrate the high level of controllability of these blue light switches. These switches are based on the utilization of blue light-sensing light-oxygen-voltage (LOV) domains from Avena sativa and Erythrobacter litoralis. The LOV domain von A. sativa, asLOV2, was engineered to cage a peptide tag which is exposed upon illumination with blue light, making the peptide tag accessible for its binding partner ePDZ. This dimerization event leads to induction of gene expression, which is fully reversible in the dark. The second blue light-regulated gene expression switch is utilizing the engineered EL222 transcription factor from E. litoralis, reduced to the minimal number of components needed for light-regulated activation: the LOV domain and a helix-turn-helix motiv. Upon blue light illumination, EL222 dimerization and DNA binding is induced. This system was constructed as a blue-on and a blue-off system, by fusing either a VP16 transactivator or a Sql1 repressor to the EL222. The functionality of both versions of this system could be demonstrated here. Besides the design and application of synthetic tools to implement highly controllable gene expression switches, another characteristic approach in synthetic biology is the usage of orthogonal systems to study complex signaling pathways. Here we report on the utilization of this approach to study the interaction of components involved in the formation of root hairs in Arabidopsis thaliana. Therefore, we heterologously expressed three key players of this pathway, GEF3, GEF4 and the small GTPase ROP2, in HeLa cells and microscopically analyzed the induced phenotype. The obtained results indicate the induction of polar cell outgrowth, induced by the interaction of plant GEF3, GEF4 and the small GTPase ROP2 with mammalian cell proteins regulating this process in mammals. These observations provide the basis for further orthogonal research on these cell outgrowth mechanisms, and their level of conservation among eukaryotes. In the course of this work, a fully equipped synthetic biology toolbox for U. maydis was implemented, proving basic researchers and biotechnologist with highly versatile tools and systems for a multitude of applications in this organism. Additionally, we discovered strong evidence for an interaction of plant and mammalian key players of polar cell outgrowth processes, indicating a higher level of functional conservation than previously expected. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie | |||||||
| Dokument erstellt am: | 21.12.2021 | |||||||
| Dateien geändert am: | 21.12.2021 | |||||||
| Promotionsantrag am: | 09.06.2020 | |||||||
| Datum der Promotion: | 13.10.2020 |
