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).

Auf dem Gelände der Landesgartenschau 2018 in Würzburg untersuchte unsere Forschungsgruppe das Anpassungsverhalten der BesucherInnen an Hitze. Ziel war es herauszufinden, wie BesucherInnen von Großveranstaltungen Hitzetage erleben und wie sie sich während unterschiedlicher Wetterbedingungen verhalten. Auf Grundlage der Ergebnisse sollen Empfehlungen zur Förderung individuellen Anpassungsverhaltens bei Hitzebelastung an Veranstalter ausgesprochen werden. An sechs aufeinanderfolgenden Wochenenden im Juli und August führten wir Temperaturmessungen, Verhaltensbeobachtungen und Befragungen unter den BesucherInnen durch. Die Wetterlage an den zwölf Erhebungstagen fiel unterschiedlich aus: Es gab sechs Hitzetage mit Temperaturen über 30 °C, vier warme Sommertage und zwei kühle Regentage. Es ließen sich unterschiedliche Anpassungsmaßnahmen bei den 2741 beobachteten BesucherInnen identifizieren. Hierzu gehören das Tragen von leichter oder kurzer Kleidung und von Kopfbedeckungen, das Mitführen von Getränken oder Schirmen sowie das Aufhalten im Schatten oder Abkühlen in einer Wasserfläche. Dabei fanden sich Unterschiede zwischen den verschiedenen Altersgruppen: Jüngere und Ältere hatten unterschiedliche Präferenzen für einzelne Anpassungsmaßnahmen. So suchten BesucherInnen über 60 Jahren bevorzugt Sitzplätze im Schatten auf, wohingegen sich Kinder zum Abkühlen in Wasserflächen aufhielten. Die Befragung von 306 BesucherInnen ergab, dass Hitzetage als stärker belastend wahrgenommen wurden als Sommer- oder Regentage. Die Mehrheit zeigte zudem ein hohes Bewusstsein für die Thematik Hitzebelastung und Anpassung. Dies spiegelte sich aber nur bei einem Teil der Befragten in ihrem tatsächlich gezeigten Anpassungsmaßnahmen wider. Offizielle Hitzewarnungen des DWD waren den meisten BesucherInnen an Tagen mit ebendiesen nicht bekannt. Auf Grundlage unserer Untersuchungsergebnisse empfehlen wir eine verbesserte Risikokommunikation in Bezug auf Hitze. Veranstalter und Behörden müssen zielgruppenspezifisch denken, wenn es um die Förderung von Hitzeanpassung geht. Angeraten werden u. a. die Schaffung von schattigen Sitzplätzen besonders für ältere BesucherInnen und Wasserstellen, an denen Kinder und Jugendliche spielen und sich erfrischen können. Da sich Hitzewellen in Zukunft häufen werden, dienen die Erkenntnisse dieser Untersuchung der Planung und Durchführung weiterer Open-Air-Veranstaltungen.

The unprecedented increase in atmospheric concentrations of carbon dioxide (CO2) and other greenhouse gases (GHG) by anthropogenic activities since the Industrial Revolution impacts on various earth system processes, commonly referred to as `climate change´ (CC). CC faces aquatic ecosystems with extreme abiotic perturbations that potentially alter the interrelations between functional autotrophic and heterotrophic plankton groups. These relations, however, modulate biogeochemical cycling and mediate the functioning of aquatic ecosystems as C sources or sinks to the atmosphere. The aim of this thesis was therefore to investigate how different aspects of CC influence community composition and functioning of pelagic heterotrophic bacteria. These organisms constitute a major component of biogeochemical cycling and largely determine the balance between autotrophic and heterotrophic processes. Due to the vast amount of potential CC impacts, this thesis focuses on the following two aspects: (1) Increased exchange of CO2 across the atmosphere-water interface and reaction of CO2 with seawater leads to profound shifts in seawater carbonate chemistry, commonly termed as `ocean acidification´ (OA), with consequences for organism physiology and the availability of dissolved inorganic carbon (DIC) in seawater. (2) The increase in atmospheric GHG concentration impacts on the efficiency with which the Earth cools to space, affecting global surface temperature and climate. With ongoing CC, shifts in frequency and severity of episodic weather events, such as storms, are expected that in particular might affect lake ecosystems by disrupting thermal summer stratification. Both aspects of CC were studied at the ecosystem-level in large-volume mesocosm experiments by using the Kiel Off-shore Mesocosms for Future Ocean Simulations (KOSMOS) deployed at different coastal marine locations, and the LakeLab facility in Lake Stechlin. We evaluated the impact of OA on heterotrophic bacterial metabolism in a brackish coastal ecosystem during low-nutrient summer months in the Baltic Sea. There are several in situ experiments that already assessed potential OA-induced changes in natural plankton communities at diverse spatial and seasonal conditions. However, most studies were performed at high phytoplankton biomass conditions, partly provoked by nutrient amendments. Our study highlights potential OA effects at low-nutrient conditions that are representative for most parts of the ocean and of particular interest in current OA research. The results suggest that during extended periods at low-nutrient concentrations, increasing pCO2 levels indirectly impact the growth balance of heterotrophic bacteria via trophic bacteria-phytoplankton interactions and shift the ecosystem to a more autotrophic system. Further work investigated how OA affects heterotrophic bacterial dissolved organic matter (DOM) transformation in two mesocsom studies, performed at different nutrient conditions. We observed similar succession patterns for individual compound pools during a phytoplankton bloom and subsequent accumulation of these compounds irrespective of the pCO2 treatment. Our results indicate that OA-induced changes in the dynamics of bacterial DOM transformation and potential impacts on DOM quality are unlikely. In addition, there have been no indications that in dependence of nutrient conditions, different amounts of photosynthetic organic matter are channelled into the more recalcitrant DOM pool. This provides novel insights into the general dynamics of the marine DOM pool. A fourth enclosure experiment in oligo-mesotrophic Lake Stechlin assessed the impact of a severe summer storm on lake bacterial communities during thermal stratification by artificially mixing. Mixing disrupted and lowered the thermocline, increasing the upper mixed layer and substantially changed water physical-chemical variables. Deep water entrainment and associated changes in water physical-chemical variables significantly affected relative bacterial abundances for about one week. Afterwards a pronounced cyanobacterial bloom developed in response to mixing which affected community assembly of heterotrophic bacteria. Colonization and mineralization of senescent phytoplankton cells by heterotrophic bacteria largely determined C-sequestration to the sediment. About six weeks after mixing, bacterial communities and measured activity parameters converged to control conditions. As such, summer storms have the potential to affect bacterial communities for a prolonged period during summer stratification. The results highlight effects on community assembly and heterotrophic bacterial metabolism that are associated to entrainment of deep water into the mixed water layer and assess consequences of an episodic disturbance event for the coupling between bacterial metabolism and autochthonous DOM production in large volume clear-water lakes. Altogether, this doctoral thesis reveales substantial sensitivities of heterotrophic bacterial metabolism and community structure in response to OA and a simulated summer storm event, which should be considered when assessing the impact of climate change on marine and lake ecosystems.

How does the international Rule of Law apply to constrain the conduct of the Executive within a constitutional State that adopts a dualist approach to the reception of international law? This paper argues that, so far from being inconsistent with the concept of the Rule of Law, the Executive within a dualist constitution has a self-enforcing obligation to abide by the obligations of the State under international law. This is not dependent on Parliament’s incorporation of treaty obligations into domestic law. It is the correlative consequence of the allocation to the Executive of the power to conduct foreign relations. The paper develops this argument in response to recent debate in the United Kingdom on whether Ministers have an obligation to comply with international law–a reference that the Government removed from the Ministerial Code. It shows that such an obligation is consistent with both four centuries of the practice of the British State and with principle.