TY - THES A1 - Pramanik, Shreya T1 - Protein reconstitution in giant vesicles N2 - Das Leben auf der Erde ist vielfältig und reicht von einzelligen Organismen bis hin zu mehrzelligen Lebewesen wie dem Menschen. Obwohl es Theorien darüber gibt, wie sich diese Organismen entwickelt haben könnten, verstehen wir nur wenig darüber, wie "Leben" aus Molekülen entstanden ist. Die synthetische Bottom-up-Biologie zielt darauf ab, minimale Zellen zu schaffen, indem sie verschiedene Module wie Kompartimentierung, Wachstum, Teilung und zelluläre Kommunikation kombiniert. Alle lebenden Zellen haben eine Membran, die sie von dem sie umgebenden wässrigen Medium trennt und sie schützt. Darüber hinaus haben alle eukaryotischen Zellen Organellen, die von intrazellulären Membranen umschlossen sind. Jede Zellmembran besteht hauptsächlich aus einer Lipiddoppelschicht mit Membranproteinen. Lipide sind amphiphile Moleküle, die molekulare Doppelschichten aus zwei Lipid-Monoschichten oder Blättchen bilden. Die hydrophoben Ketten der Lipide sind einander zugewandt, während ihre hydrophilen Kopfgruppen die Grenzflächen zur wässrigen Umgebung bilden. Riesenvesikel sind Modellmembransysteme, die Kompartimente mit einer Größe von mehreren Mikrometern bilden und von einer einzigen Lipiddoppelschicht umgeben sind. Die Größe der Riesenvesikel ist mit der Größe von Zellen vergleichbar und macht sie zu guten Membranmodellen, die mit einem Lichtmikroskop untersucht werden können. Allerdings fehlen den Riesenvesikelmembranen nach der ersten Präparation Membranproteine, die in weiteren Präparationsschritten in diese Membranen eingebaut werden müssen. Je nach Protein kann es entweder über Ankerlipide an eines der Membranblättchen gebunden oder über seine Transmembrandomänen in die Lipiddoppelschicht eingebaut werden. Diese Arbeit befasst sich mit der Herstellung von Riesenvesikeln und der Rekonstitution von Proteinen in diesen Vesikeln. Außerdem wird ein mikrofluidischer Chip entworfen, der in verschiedenen Experimenten verwendet werden kann. Die Ergebnisse dieser Arbeit werden anderen Forschern helfen, die Protokolle für die Herstellung von GUVs zu verstehen, Proteine in GUVs zu rekonstituieren und Experimente mit dem mikrofluidischen Chip durchzuführen. Auf diese Weise wird die vorliegende Arbeit für das langfristige Ziel von Nutzen sein, die verschiedenen Module der synthetischen Biologie zu kombinieren, um eine Minimalzelle zu schaffen. N2 - Life on Earth is diverse and ranges from unicellular organisms to multicellular creatures like humans. Although there are theories about how these organisms might have evolved, we understand little about how ‘life’ started from molecules. Bottom-up synthetic biology aims to create minimal cells by combining different modules, such as compartmentalization, growth, division, and cellular communication. All living cells have a membrane that separates them from the surrounding aqueous medium and helps to protect them. In addition, all eukaryotic cells have organelles that are enclosed by intracellular membranes. Each cellular membrane is primarily made of a lipid bilayer with membrane proteins. Lipids are amphiphilic molecules that assemble into molecular bilayers consisting of two leaflets. The hydrophobic chains of the lipids in the two leaflets face each other, and their hydrophilic headgroups face the aqueous surroundings. Giant unilamellar vesicles (GUVs) are model membrane systems that form large compartments with a size of many micrometers and enclosed by a single lipid bilayer. The size of GUVs is comparable to the size of cells, making them good membrane models which can be studied using an optical microscope. However, after the initial preparation, GUV membranes lack membrane proteins which have to be reconstituted into these membranes by subsequent preparation steps. Depending on the protein, it can be either attached via anchor lipids to one of the membrane leaflets or inserted into the lipid bilayer via its transmembrane domains. The first step is to prepare the GUVs and then expose them to an exterior solution with proteins. Various protocols have been developed for the initial preparation of GUVs. For the second step, the GUVs can be exposed to a bulk solution of protein or can be trapped in a microfluidic device and then supplied with the protein solution. To minimize the amount of solution and for more precise measurements, I have designed a microfluidic device that has a main channel, and several dead-end side channels that are perpendicular to the main channel. The GUVs are trapped in the dead-end channels. This design exchanges the solution around the GUVs via diffusion from the main channel, thus shielding the GUVs from the flow within the main channel. This device has a small volume of just 2.5 μL, can be used without a pump and can be combined with a confocal microscope, enabling uninterrupted imaging of the GUVs during the experiments. I used this device for most of the experiments on GUVs that are discussed in this thesis. In the first project of the thesis, a lipid mixture doped with an anchor lipid was used that can bind to a histidine chain (referred to as His-tag(ged) or 6H) via the metal cation Ni2+. This method is widely used for the biofunctionalization of GUVs by attaching proteins without a transmembrane domain. Fluorescently labeled His-tags which are bound to a membrane can be observed in a confocal microscope. Using the same lipid mixture, I prepared the GUVs with different protocols and investigated the membrane composition of the resulting GUVs by evaluating the amount of fluorescently labeled His-tagged molecules bound to their membranes. I used the microfluidic device described above to expose the outer leaflet of the vesicle to a constant concentration of the His-tagged molecules. Two fluorescent molecules with a His-tag were studied and compared: green fluorescent protein (6H-GFP) and fluorescein isothiocyanate (6H-FITC). Although the quantum yield in solution is similar for both molecules, the brightness of the membrane-bound 6H-GFP is higher than the brightness of the membrane-bound 6H-FITC. The observed difference in the brightness reveals that the fluorescence of the 6H-FITC is quenched by the anchor lipid via the Ni2+ ion. Furthermore, my measurements also showed that the fluorescence intensity of the membranebound His-tagged molecules depends on microenvironmental factors such as pH. For both 6H-GFP and 6H-FITC, the interaction with the membrane is quantified by evaluating the equilibrium dissociation constant. The membrane fluorescence is measured as a function of the fluorophores’ molar concentration. Theoretical analysis of these data leads to the equilibrium dissociation constants of (37.5 ± 7.5) nM for 6H-GFP and (18.5 ± 3.7) nM for 6H-FITC. The anchor lipid mentioned previously used the metal cation Ni2+ to mediate the bond between the anchor lipid and the His-tag. The Ni2+ ion can be replaced by other transition metal ions. Studies have shown that Co3+ forms the strongest bonds with the His-tags attached to proteins. In these studies, strong oxidizing agents were used to oxidize the Co2+ mediated complex with the His-tagged protein to a Co3+ mediated complex. This procedure puts the proteins at risk of being oxidized as well. In this thesis, the vesicles were first prepared with anchor lipids without any metal cation. The Co3+ was added to these anchor lipids and finally the His-tagged protein was added to the GUVs to form the Co3+ mediated bond. This system was also established using the microfluidic device. The different preparation procedures of GUVs usually lead to vesicles with a spherical morphology. On the other hand, many cell organelles have a more complex architecture with a non spherical topology. One fascinating example is provided by the endoplasmic reticulum (ER) which is made of a continuous membrane and extends throughout the cell in the form of tubes and sheets. The tubes are connected by three-way junctions and form a tubular network of irregular polygons. The formation and maintenance of these reticular networks requires membrane proteins that hydrolyize guanosine triphosphate (GTP). One of these membrane proteins is atlastin. In this thesis, I reconstituted the atlastin protein in GUV membranes using detergent-assisted reconstitution protocols to insert the proteins directly into lipid bilayers. This thesis focuses on protein reconstitution by binding His-tagged proteins to anchor lipids and by detergent-assisted insertion of proteins with transmembrane domains. It also provides the design of a microfluidic device that can be used in various experiments, one example is the evaluation of the equilibrium dissociation constant for membrane-protein interactions. The results of this thesis will help other researchers to understand the protocols for preparing GUVs, to reconstitute proteins in GUVs, and to perform experiments using the microfluidic device. This knowledge should be beneficial for the long-term goal of combining the different modules of synthetic biology to make a minimal cell. KW - protein reconstitution KW - giant vesicles KW - microfluidics KW - synthetic biology KW - Riesenvesikel KW - Mikrofluidik KW - Proteinrekonstitution KW - synthetische Biologie Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-612781 ER - TY - THES A1 - Demin, Paul T1 - Blaulicht-aktivierbares Proteinexpressionssystem in Saccharomyces cerevisiae T1 - Blue light-inducible protein expression system in Saccharomyces cerevisiae N2 - Synthetische Transkriptionsfaktoren bestehen wie natürliche Transkriptionsfaktoren aus einer DNA-Bindedomäne, die sich spezifisch an die Bindestellensequenz vor dem Ziel-Gen anlagert, und einer Aktivierungsdomäne, die die Transkriptionsmaschinerie rekrutiert, sodass das Zielgen exprimiert wird. Der Unterschied zu den natürlichen Transkriptionsfaktoren ist, sowohl dass die DNA-Bindedomäne als auch die Aktivierungsdomäne wirtsfremd sein können und dadurch künstliche Stoffwechselwege im Wirt, größtenteils chemisch, induziert werden können. Optogenetische synthetische Transkriptionsfaktoren, die hier entwickelt wurden, gehen einen Schritt weiter. Dabei ist die DNA-Bindedomäne nicht mehr an die Aktivierungsdomäne, sondern mit dem Blaulicht-Photorezeptor CRY2 gekoppelt. Die Aktivierungsdomäne wurde mit dem Interaktionspartner CIB1 fusioniert. Unter Blaulichtbestrahlung dimerisieren CRY2 und CIB1 und damit einhergehend die beiden Domänen, sodass ein funktionsfähiger Transkriptionsfaktor entsteht. Dieses System wurde in die Saccharomyces cerevisiae genomisch integriert. Verifiziert wurde das konstruierte System mit Hilfe des Reporters yEGFP, welcher durchflusszytometrisch detektiert werden konnte. Es konnte gezeigt werden, dass die yEGFP Expression variabel gestaltet werden kann, indem unterschiedlich lange Blaulichtimpulse ausgesendet wurden, die DNA-Bindedomäne, die Aktivierungsdomäne oder die Anzahl der Bindestellen, an dem sich die DNA-Bindedomäne anlagert, verändert wurden. Um das System für industrielle Anwendungen attraktiv zu gestalten, wurde das System vom Deepwell-Maßstab auf Photobioreaktor-Maßstab hochskaliert. Außerdem erwies sich das Blaulichtsystem sowohl im Laborstamm YPH500 als auch im industriell oft verwendeten Hefestamm CEN.PK als funktional. Des Weiteren konnte ein industrierelevante Protein ebenso mit Hilfe des verifizierten Systems exprimiert werden. Schlussendlich konnte in dieser Arbeit das etablierte Blaulicht-System erfolgreich mit einem Rotlichtsystem kombiniert werden, was zuvor noch nicht beschrieben wurde. N2 - Like natural transcription factors, synthetic transcription factors consist of a DNA-binding domain that attaches specifically to the binding site sequence in front of the target gene, and an activation domain that recruits the transcription machinery so that the target gene is expressed. The difference to natural transcription factors is that both the DNA binding domain and the activation domain can be host foreign and artificial metabolic pathways, mostly chemically, can be induced in the host. In this work, new optogenetic synthetic transcription factors were developed so that chemical induction becomes obsolete. The DNA binding domain is no longer linked to the activation domain but to the blue light photoreceptor CRY2. The activation domain was fused to the interaction partner CIB1. Upon blue light irradiation, CRY2 and CIB1 dimerize and thus the two domains, resulting in a functional transcription factor. Six different prokaryotic DNA-binding domains and a total of two different activation domains, viral and fungal, were recombined with CRY2 and CIB1, respectively, and genomically integrated into the eukaryotic cell factory Saccharomyces cerevisiae. Since the blue light dimerization is based on the chromophore FAD, which the yeast can synthesize itself, only the blue light had to be switched on for the induction. The constructed system was verified with the help of the reporter yEGFP, which could be detected by flow cytometry. It could be shown that the yEGFP expression could be made variable by emitting blue light pulses of different lengths, changing the DNA binding domain, the activation domain or the copy number of binding sites at which the DNA binding domain attaches. To make the system attractive for industrial applications, the system was scaled up from deepwell scale to photobioreactor scale. In addition, the blue light system proved to be functional both in the laboratory strain YPH500 and in the yeast strain CEN.PK, which is often used industrially. Furthermore, the industrially relevant protein VP1 could also be expressed using the verified system. Due to its great flexibility, the blue light system established here was christened/named FLIRT (Flexible Blue Light Induced Transcription). Finally, in this work, the established flirt system could be successfully combined with a red light system, which has not been described before. KW - Synthetische Biologie KW - Hefe KW - Saccharomyces cerevisiae KW - Blaulicht KW - Transkriptionsfaktoren KW - Bioreaktor KW - bioreactor KW - blue light KW - budding yeast KW - Saccharomyces cerevisiae KW - synthetic biology KW - transcription factors Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-559696 ER - TY - THES A1 - Chandrakanth Shetty, Sunidhi T1 - Directed chemical communication in artificial eukaryotic cells T1 - Gezielte chemische Kommunikation in künstlichen eukaryotischen Zellen N2 - Eukaryotic cells can be regarded as complex microreactors capable of performing various biochemical reactions in parallel which are necessary to sustain life. An essential prerequisite for these complex metabolic reactions to occur is the evolution of lipid membrane-bound organelles enabling compartmental- ization of reactions and biomolecules. This allows for a spatiotemporal control over the metabolic reactions within the cellular system. Intracellular organi- zation arising due to compartmentalization is a key feature of all living cells and has inspired synthetic biologists to engineer such systems with bottom-up approaches. Artificial cells provide an ideal platform to isolate and study specific re- actions without the interference from the complex network of biomolecules present in biological cells. To mimic the hierarchical architecture of eukaryotic cells, multi-compartment assemblies with nested liposomal structures also re- ferred to as multi-vesicular vesicles (MVVs) have been widely adopted. Most of the previously reported multi-compartment systems adopt bulk method- ologies which suffer from low yield and poor control over size. Microfluidic strategies help circumvent these issues and facilitate a high-throughput and robust technique to assemble MVVs of uniform size distribution. In this thesis, firstly, the bulk methodologies are explored to build MVVs and implement a synthetic signalling cascade. Next, a polydimethylsiloxane (PDMS)-based microfluidic platform is introduced to build MVVs and the significance of PEGylated lipids for the successful encapsulation of inner com- partments to generate stable multi-compartment systems is highlighted. Next, a novel two-inlet channel PDMS-based microfluidic device to create MVVs encompassing a three-step enzymatic reaction cascade is presented. A directed reaction pathway comprising of the enzymes α-glucosidase (α-Glc), glucose oxidase (GOx), and horseradish peroxidase (HRP) spanning across three compartments via reconstitution of size-selective membrane proteins is described. Furthermore, owing to the monodispersity of our MVVs due to microfluidic strategies, this platform is employed to study the effect of com- partmentalization on reaction kinetics. Further integration of cell-free expression module into the MVVs would allow for gene-mediated signal transduction within artificial eukaryotic cells. Therefore, the chemically inducible cell-free expression of a membrane protein alpha-hemolysin and its further reconstitution into liposomes is carried out. In conclusion, the present thesis aims to build artificial eukaryotic cells to achieve size-selective chemical communication that also show potential for applications as micro reactors and as vehicles for drug delivery. N2 - Eukaryontische Zellen können als komplexe Mikroreaktoren betrachtet werden, die in der Lage sind, verschiedene biochemische Reaktionen parallel durchzuführen, die für die Aufrechterhaltung des Lebens notwendig sind. Eine wesentliche Voraussetzung für die Durchführung dieser komplexen Stoffwechselreaktionen ist die Entwicklung von Organellen mit Lipidmembranen, die eine Kompartimentierung von Reaktionen und Biomolekülen ermöglichen. Dies ermöglicht eine räumlich-zeitliche Kontrolle über die Stoffwechselreaktionen innerhalb des zellulären Systems. Die durch die Kompartimentierung entstehende intrazelluläre Organisation ist ein Schlüsselmerkmal aller lebenden Zellen und hat synthetische Biologen dazu inspiriert, solche Systeme mit Bottom-up-Ansätzen zu entwickeln. Künstliche Zellen bieten eine ideale Plattform, um spezifische Reaktionen zu isolieren und zu untersuchen, ohne dass das komplexe Netzwerk von Biomolekülen, das in biologischen Zellen vorhanden ist, stört. Um die hierarchische Architektur eukaryontischer Zellen zu imitieren, haben sich Multikompartiment-Anordnungen mit verschachtelten liposomalen Strukturen, die auch als multivesikuläre Vesikel (MVV) bezeichnet werden, durchgesetzt. Die meisten der bisher vorgestellten Multikompartiment-Systeme basieren auf Bulk-Methoden, die eine geringe Ausbeute und eine schlechte Kontrolle über die Größe aufweisen. Mikrofluidische Strategien helfen, diese Probleme zu umgehen und ermöglichen eine robuste Technik mit hohem Durchsatz, um MVVs mit einheitlicher Größenverteilung herzustellen. In dieser Dissertation werden zunächst die Bulk-Methoden zum Aufbau von MVVs und zur Implementierung einer synthetischen Signalkaskade untersucht. Anschließend wird eine auf Polydimethylsiloxan (PDMS) basierende mikrofluidische Plattform zur Herstellung von MVVs vorgestellt und die Bedeutung von PEGylierten Lipiden für die erfolgreiche Verkapselung der inneren Kompartimente zur Erzeugung stabiler Multikompartiment-Systeme hervorgehoben. Es wird ein neuartiges mikrofluidisches Gerät mit zwei Einlasskanälen auf PDMS-Basis zur Herstellung von MVVs vorgestellt, das eine dreistufige enzymatische Reaktionskaskade umfasst. Es wird ein gerichteter Reaktionsweg beschrieben, der die Enzyme α-Glucosidase (α-Glc), Glucoseoxidase (GOx) und Meerrettichperoxidase (HRP) umfasst und sich über drei Kompartimente erstreckt, die durch die Rekonstitution von größenselektiven Membranproteinen entstehen. Aufgrund der Monodispersität unserer MVVs durch mikrofluidische Strategien nutze ich diese Plattform außerdem, um die Auswirkungen der Kompartimentierung auf die Reaktionskinetik zu untersuchen. Eine weitere Integration von zellfreien Expressionsmodulen in MVVs würde eine genvermittelte Signaltransduktion in künstlichen eukaryotischen Zellen ermöglichen. Daher wird die chemisch induzierbare zellfreie Expression eines Membranproteins alpha-Hämolysin und seine weitere Rekonstitution in Liposomen durchgeführt. Zusammenfassend lässt sich sagen, dass die vorliegende Arbeit darauf abzielt, künstliche eukaryotische Zellen zu bauen, um eine größenselektive chemische Kommunikation zu erreichen, und das Potenzial für Anwendungen als Mikroreaktoren und als Vehikel für die Verabreichung von Medikamenten aufweisen. KW - microfluidics KW - synthetic biology KW - Mikrofluidik KW - synthetische Biologie Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-533642 ER - TY - THES A1 - Kirchhofer, Tabea T1 - The development of multi - compartmentalised systems for the directed organisation of artificial cells N2 - Membrane contact sites are of particular interest in the field of synthetic biology and biophysics. They are involved in a great variety of cellular functions. They form in between two cellular organelles or an organelle and the plasma membrane in order to establish a communication path for molecule transport or signal transmission. The development of an artificial membrane system which can mimic membrane contact sites using bottom up synthetic biology was the goal of this research study. For this, a multi - compartmentalised giant unilamellar vesicle (GUV) system was created with the membrane of the outer vesicle mimicking the plasma membrane and the inner GUVs posing as cellular organelles. In the following steps, three different strategies were used to achieve an internal membrane - membrane adhesion. N2 - Viele bedeutende Prozesse einer Zelle spielen sich an den Berührungsstellen zwischen Zellmembranen und auch zwischen Zellmembranen und der Plasmamembran ab. An diesen, aus spezifischen Lipiden und Proteinen aufgebauten Kontaktstellen, können auf Grund der geringen Entfernung Signale und auch Moleküle ausgetauscht werden. Ziel dieses Forschungsprojektes war die Entwicklung eines künstlichen Zellmembransystems, das in der Lage ist diese Kontaktstellen nachzubilden. Dafür wurden multikompartmentalisierte riesige unilamellare Vesikel (GUVs) aufgebaut. Dies bedeutet, dass sich ein GUV innerhalb eines anderen GUVs befindet. Das äußere Vesikel bildet in diesem System die Plasma Membran, während das Innere als Zellorganelle fungiert. Dieses System wird auch als Vesosom bezeichnet. Im Folgenden wurden drei verschiedene Strategien entwickelt, um interne Haftung (Adhäsion) zwischen den Membranen zu erzeugen. KW - vesicle studies KW - membrane science KW - synthetic biology KW - internal membrane-membrane adhesion KW - artificial cells KW - multi-compartmentalised vesicles KW - künstliche Zellen KW - interne Membran-Membran Adhäsion KW - Membranforschung bzw. Membranwissenschaften KW - multi-kompartmentalisierte Vesikel KW - Synthetische Biologie KW - Vesikel Forschung/Vesikel Studien Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-528428 ER - TY - JOUR A1 - Brechun, Katherine Emily A1 - Arndt, Katja Maren A1 - Woolley, G. Andrew T1 - Selection of protein-protein interactions of desired affinities with a bandpass circuit JF - Journal of molecular biology : JMB N2 - We have developed a genetic circuit in Escherichia coli that can be used to select for protein-protein interactions of different strengths by changing antibiotic concentrations in the media. The genetic circuit links protein-protein interaction strength to beta-lactamase activity while simultaneously imposing tuneable positive and negative selection pressure for beta-lactamase activity. Cells only survive if they express interacting proteins with affinities that fall within set high- and low-pass thresholds; i.e. the circuit therefore acts as a bandpass filter for protein-protein interactions. We show that the circuit can be used to recover protein-protein interactions of desired affinity from a mixed population with a range of affinities. The circuit can also be used to select for inhibitors of protein-protein interactions of defined strength. (C) 2018 Elsevier Ltd. All rights reserved. KW - synthetic biology KW - genetic circuit KW - biological engineering KW - protein-protein interactions KW - twin-arginine translocation KW - selection system Y1 - 2018 U6 - https://doi.org/10.1016/j.jmb.2018.11.011 SN - 0022-2836 SN - 1089-8638 VL - 431 IS - 2 SP - 391 EP - 400 PB - Elsevier CY - London ER - TY - JOUR A1 - de la Cruz, Jorge Gonzalez A1 - Machens, Fabian A1 - Messerschmidt, Katrin A1 - Bar-Even, Arren T1 - Core Catalysis of the Reductive Glycine Pathway Demonstrated in Yeast JF - ACS synthetic biology N2 - One-carbon (C1) compounds are attractive microbial feedstocks as they can be efficiently produced from widely available resources. Formate, in particular, represents a promising growth substrate, as it can be generated from electrochemical reduction of CO2 and fed to microorganisms in a soluble form. We previously identified the synthetic reductive glycine pathway as the most efficient route for aerobic growth on formate. We further demonstrated pathway activity in Escherichia coli after expression of both native and foreign genes. Here, we explore whether the reductive glycine pathway could be established in a model microorganism using only native enzymes. We used the yeast Saccharomyces cerevisiae as host and show that overexpression of only endogenous enzymes enables glycine biosynthesis from formate and CO2 in a strain that is otherwise auxotrophic for glycine. We find the pathway to be highly active in this host, where 0.125 mM formate is sufficient to support growth. Notably, the formate-dependent growth rate of the engineered S. cerevisiae strain remained roughly constant over a very wide range of formate concentrations, 1-500 mM, indicating both high affinity for formate use and high tolerance toward elevated concentration of this C1 feedstock. Our results, as well the availability of endogenous NAD-dependent formate dehydrogenase, indicate that yeast might be an especially suitable host for engineering growth on formate. KW - metabolic engineering KW - synthetic biology KW - one-carbon metabolism KW - carbon labeling KW - tetrahydrofolate KW - glycine cleavage system Y1 - 2019 U6 - https://doi.org/10.1021/acssynbio.8b00464 SN - 2161-5063 VL - 8 IS - 5 SP - 911 EP - 917 PB - American Chemical Society CY - Washington ER - TY - GEN A1 - Lukan, Tjaša A1 - Machens, Fabian A1 - Coll, Anna A1 - Baebler, Špela A1 - Messerschmidt, Katrin A1 - Gruden, Kristina T1 - Plant X-tender BT - an extension of the AssemblX system for the assembly and expression of multigene constructs in plants T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scarfree and sequence-independent multigene assembly strategy AssemblX,based on overlapdepended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 990 KW - ligation cloning extract KW - DNA cloning KW - synthetic biology KW - multiple genes KW - vector system KW - transformation KW - recombination KW - protein KW - RNA KW - Methylation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-446281 SN - 1866-8372 IS - 990 ER - TY - JOUR A1 - Naseri, Gita A1 - Balazadeh, Salma A1 - Machens, Fabian A1 - Kamranfar, Iman A1 - Messerschmidt, Katrin A1 - Müller-Röber, Bernd T1 - Plant-Derived Transcription Factors for Orthologous Regulation of Gene Expression in the Yeast Saccharomyces cerevisiae JF - ACS synthetic biology N2 - Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harboring cognate cis regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver/reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC-EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF-DNA binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast. KW - Arabidopsis thaliana KW - artificial transcription factor KW - NAC transcription factor KW - synthetic biology KW - plant Y1 - 2017 U6 - https://doi.org/10.1021/acssynbio.7b00094 SN - 2161-5063 VL - 6 SP - 1742 EP - 1756 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Gorochowski, Thomas E. A1 - Aycilar-Kucukgoze, Irem A1 - Bovenberg, Roel A. L. A1 - Roubos, Johannes A. A1 - Ignatova, Zoya T1 - A Minimal Model of Ribosome Allocation Dynamics Captures Trade-offs in Expression between Endogenous and Synthetic Genes JF - ACS synthetic biology N2 - Cells contain a finite set of resources that must be distributed across many processes to ensure survival. Among them, the largest proportion of cellular resources is dedicated to protein translation. Synthetic biology often exploits these resources in executing orthogonal genetic circuits, yet the burden this places on the cell is rarely considered. Here, we develop a minimal model of ribosome allocation dynamics capturing the demands on translation when expressing a synthetic construct together with endogenous genes required for the maintenance of cell physiology. Critically, it contains three key variables related to design parameters of the synthetic construct covering transcript abundance, translation initiation rate, and elongation time. We show that model-predicted changes in ribosome allocation closely match experimental shifts in synthetic protein expression rate and cellular growth. Intriguingly, the model is also able to accurately infer transcript levels and translation times after further exposure to additional ambient stress. Our results demonstrate that a simple model of resource allocation faithfully captures the redistribution of protein synthesis resources when faced with the burden of synthetic gene expression and environmental stress. The tractable nature of the model makes it a versatile tool for exploring the guiding principles of efficient heterologous expression and the indirect interactions that can arise between synthetic circuits and their host chassis because of competition for shared translational resources. KW - protein biosynthesis KW - translation KW - synthetic biology KW - systems biology Y1 - 2016 U6 - https://doi.org/10.1021/acssynbio.6b00040 SN - 2161-5063 VL - 5 SP - 710 EP - 720 PB - American Chemical Society CY - Washington ER - TY - THES A1 - Naseri, Gita T1 - Plant-derived transcription factors and their application for synthetic biology approaches in Saccharomyces cerevisiae T1 - Pflanzenbasierte Transkriptionsfaktoren und ihre Anwendungen in der synthetischen Biologie in Saccharomyces cerevisiae N2 - Bereits seit 9000 Jahren verwendet die Menschheit die Bäckerhefe Saccharomyces cerevisiae für das Brauen von Bier, aber erst seit 150 Jahren wissen wir, dass es sich bei diesem unermüdlichen Helfer im Brauprozess um einzellige, lebende Organismen handelt. Und die Bäckerhefe kann noch viel mehr. Im Rahmen des Forschungsgebietes der Synthetischen Biologie soll unter anderem die Bäckerhefe als innovatives Werkzeug für die biobasierte Herstellung verschiedenster Substanzen etabliert werden. Zu diesen Substanzen zählen unter anderem Feinchemikalien, Biokraftstoffe und Biopolymere sowie pharmakologisch und medizinisch interessante Pflanzenstoffe. Damit diese verschiedensten Substanzen in der Bäckerhefe hergestellt werden können, müssen große Mengen an Produktionsinformationen zum Beispiel aus Pflanzen in die Hefezellen übertragen werden. Darüber hinaus müssen die neu eingebrachten Biosynthesewege reguliert und kontrolliert in den Zellen ablaufen. Auch Optimierungsprozesse zur Erhöhung der Produktivität sind notwendig. Für alle diese Arbeitsschritte mangelt es bis heute an anwendungsbereiten Technologien und umfassenden Plattformen. Daher wurden im Rahmen dieser Doktorarbeit verschiedene Technologien und Plattformen zur Informationsübertragung, Regulation und Prozessoptimierung geplant und erzeugt. Für die Konstruktion von Biosynthesewegen in der Bäckerhefe wurde als erstes eine Plattform aus neuartigen Regulatoren und Kontrollelementen auf der Basis pflanzlicher Kontrollelemente generiert und charakterisiert. Im zweiten Schritt erfolgte die Entwicklung einer Technologie zur kombinatorischen Verwendung der Regulatoren in der Planung und Optimierung von Biosynthesewegen (COMPASS). Abschließend wurde eine Technologie für die Prozessoptimierung der veränderten Hefezellen entwickelt (CapRedit). Die Leistungsfähigkeit der entwickelten Plattformen und Technologien wurde durch eine Optimierung der Produktion von Carotenoiden (Beta-Carotin und Beta-Ionon) und Flavonoiden (Naringenin) in Hefezellen nachgewiesen. Die im Rahmen der Arbeit etablierten neuartigen Plattformen und innovativen Technologien sind ein wertvoller Grundbaustein für die Erweiterung der Nutzbarkeit der Bäckerhefe. Sie ermöglichen den Einsatz der Hefezellen in kosteneffizienten Produktionswegen und alternativen chemischen Wertschöpfungsketten. Dadurch können zum Beispiel Biokraftstoffe und pharmakologisch interessante Pflanzenstoffe unter Verwendung von nachwachsenden Rohstoffen, Reststoffen und Nebenprodukten hergestellt werden. Darüber hinaus ergeben sich Anwendungsmöglichkeiten zur Bodensanierung und Wasseraufbereitung. N2 - Plant-derived Transcription Factors for Orthologous Regulation of Gene Expression in the Yeast Saccharomyces cerevisiae Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects where tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harbouring cognate cis-regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver / reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast, than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC - EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF – DNA-binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast. COMPASS: Rapid combinatorial optimization of biochemical pathways based on artificial transcription factors We established a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) for controlling the expression of pathway genes, and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, we equipped COMPASS with multi-locus CRISPR/Cas9-mediated modification capacity. In its current realization, COMPASS allows combinatorial optimization of up to ten pathway genes, each transcriptionally controlled by nine different ATFs spanning a 10-fold difference in expression strength. The application of COMPASS was demonstrated by generating cell libraries producing beta-carotene and co-producing beta-ionone and biosensor-responsive naringenin. COMPASS will have many applications in other synthetic biology projects that require gene expression balancing. CaPRedit: Genome editing using CRISPR-Cas9 and plant-derived transcriptional regulators for the redirection of flux through the FPP branch-point in yeast. Technologies developed over the past decade have made Saccharomyces cerevisiae a promising platform for production of different natural products. We developed CRISPR/Ca9- and plant derived regulator-mediated genome editing approach (CaPRedit) to greatly accelerate strain modification and to facilitate very low to very high expression of key enzymes using inducible regulators. CaPRedit can be implemented to enhance the production of yeast endogenous or heterologous metabolites in the yeast S. cerevisiae. The CaPRedit system aims to faciltiate modification of multiple targets within a complex metabolic pathway through providing new tools for increased expression of genes encoding rate-limiting enzymes, decreased expression of essential genes, and removed expression of competing pathways. This approach is based on CRISPR/Cas9-mediated one-step double-strand breaks to integrate modules containing IPTG-inducible plant-derived artificial transcription factor and promoter pair(s) in a desired locus or loci. Here, we used CaPRedit to redirect the yeast endogenous metabolic flux toward production of farnesyl diphosphate (FPP), a central precursor of nearly all yeast isoprenoid products, by overexpression of the enzymes lead to produce FPP from glutamate. We found significantly higher beta-carotene accumulation in the CaPRedit-mediated modified strain than in the wild type (WT) strain. More specifically, CaPRedit_FPP 1.0 strain was generated, in which three genes involved in FPP synthesis, tHMG1, ERG20, and GDH2, were inducibly overexpressed under the control of strong plant-derived ATFPs. The beta–carotene accumulated in CaPRedit_FPP 1.0 strain to a level 1.3-fold higher than the previously reported optimized strain that carries the same overexpressed genes (as well as additional genetic modifications to redirect yeast endogenous metabolism toward FPP production). Furthermore, the genetic modifications implemented in CaPRedit_FPP 1.0 strain resulted in only a very small growth defect (growth rate relative to the WT is ~ -0.03). KW - synthetic biology KW - Saccharomyces cerevisiae KW - artificial transcription factor KW - combinatorial optimization KW - biosensor KW - DNA assembly KW - pathway engineering KW - artifizielle Transkriptionsfaktoren KW - Biosensor KW - kombinatorische Optimierung KW - DNA assembly KW - Saccharomyces cerevisiae KW - synthetische Biologie KW - pathway engineering Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-421514 ER -