TY - JOUR A1 - Zhang, Youjun A1 - Sampathkumar, Arun A1 - Kerber, Sandra Mae-Lin A1 - Swart, Corné A1 - Hille, Carsten A1 - Seerangan, Kumar A1 - Graf, Alexander A1 - Sweetlove, Lee A1 - Fernie, Alisdair R. T1 - A moonlighting role for enzymes of glycolysis in the co-localization of mitochondria and chloroplasts JF - Nature Communications N2 - Glycolysis is one of the primordial pathways of metabolism, playing a pivotal role in energy metabolism and biosynthesis. Glycolytic enzymes are known to form transient multi-enzyme assemblies. Here we examine the wider protein-protein interactions of plant glycolytic enzymes and reveal a moonlighting role for specific glycolytic enzymes in mediating the co-localization of mitochondria and chloroplasts. Knockout mutation of phosphoglycerate mutase or enolase resulted in a significantly reduced association of the two organelles. We provide evidence that phosphoglycerate mutase and enolase form a substrate-channelling metabolon which is part of a larger complex of proteins including pyruvate kinase. These results alongside a range of genetic complementation experiments are discussed in the context of our current understanding of chloroplast-mitochondrial interactions within photosynthetic eukaryotes. Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-14812 SN - 2041-1723 VL - 11 SP - 4509 ER - TY - JOUR A1 - Wieland, Ralf A1 - Kuhls, Katrin A1 - Lentz, Hartmut H.K. A1 - Conraths, Franz A1 - Kampen, Helge A1 - Werner, Doreen T1 - Combined climate and regional mosquito habitat model based on machine learning JF - Ecological Modelling N2 - Besides invasive mosquito species also several native species are proven or suspected vectors of arboviruses as West Nile or Usutu virus in Western Europe. Habitat models of these native vectors can be a helpful tool for assessing the risk of autochthonous occurrence, outbreaks and spread of diseases caused by such arboviruses. Modelling native mosquitoes is complicated because of the perfect adaptation to the climatic and landscape conditions and their high abundance in contrast to invasive species. Here we present a new approach for such a habitat model for native mosquito species in Germany, which are considered as vectors of West Nile virus (WNV). Epizootic emergence of WNV was registered in Germany since 2018. The models are based on surveillance data of mosquitoes from the German citizen science project “Mückenatlas” complemented by data from systematic trap monitoring in Germany, and on data freely available from the Deutscher Wetterdienst (DWD) and OpenStreetMap (OSM). While climatic factors still play an important role, we could show that habitat suitability is predictable only by the combination of the climate model with a regional model. Both models were based on a machine-learning approach using XGBoost. Evaluation of the accuracy of the models was done by statistical analysis, determining among others feature importances using the SHAP-Library. Final output of the combined climatic and regional models are maps showing the superposed habitat suitability which are generated through a number of steps described in detail. These maps also include the registered cases of WNV infections in the selected region of Germany. KW - citizen science data KW - mosquito habitat modelling KW - machine learning KW - XGBoost KW - West Nile virus KW - vector borne disease Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-15649 SN - 0304-3800 VL - 452 CY - Elsevier ER - TY - GEN A1 - Weinert, Stefan A1 - Wohlfahrt, Ingolf A1 - Schmid, Andrea A1 - Frohme, Marcus T1 - Einführung eines Qualitätsmanagement-Systems in einem molekularbiologischen Labor einer Hochschule T2 - Wissenschaftliche Beiträge 2009/2010 N2 - Zertifizierte Qualitätsmanagementsysteme (QM-Systeme) sind in akademischen Forschungslaboren bisher kaum vertreten. Hier ist vor allem die Bedeutung eines zertifizierten Labors vor dem Hintergrund der Veröffentlichung wissenschaftlicher Publikationen scheinbar bedeutungslos. Im industriellen Bereich hingegen sind QM-Systeme weitaus häufiger eingeführt. Labore im akademischen Bereich stehen jedoch grundsätzlich in der Verantwortung, schonend mit den Ressourcen des Staates umzugehen, und haben darüber hinaus das Bestreben, Forschungs-/Industrieaufträge einzuwerben. Mit dieser Publikation wird gezeigt, wie durch die Einrichtung eines QM-Systems und der Einhaltung der normativen Vorgabe ein verbesserter schonender Umgang mit staatlichen Ressourcen möglich ist, die Ergebnissicherheit erhöht, Betriebsabläufe optimiert, die Chancen für Studenten auf dem Arbeitsmarkt verbessert werden und mehr Forschungs-/Industrieaufträge akquiriert werden können. Es wird dargestellt, wie ein QM-System in einem Labor eingerichtet werden kann. Dabei wird erläutert, welche QM-Regelwerke für ein Labor im Hochschulbereich in Betracht kommen. Zudem wird eine Methodik in Form eines Projektplans vorgestellt, mit dem die Anforderungen der Norm strukturiert umgesetzt werden können. Das QM-System wurde im Labor für Molekularbiologie und Funktionelle Genomik an der Technischen Hochschule Wildau [FH] eingeführt und im Rahmen der Zertifizierung der Hochschule in das Audit einbezogen. N2 - Up to now certified Quality-Management-Systems(QM-system) are hardly prevalent in university research laboratories. Even if a laboratory is certified, it seems to have no meaning for a scientific publication originating from this laboratory. In the industrial sector QM-systems are a lot more common. Basically, academic laboratories have to use their rescources, provided primarily by govermental funding, very sparingly and responsible. Furthermore universities have a strong endeavour to accquire funding for further research. We demonstrate here, how the establishment of a QM-system, and compliance to normative requirements, can improve the economic use of governmental funding. Furthermore a QM-system can ensure result reliability and work routines can be optimized. In addition the job perspectives for graduates as well as the possibilities to aquire funding will be improved. We present how a QM-system can be established in a lifescience- laboratory. Thereby it will be outlined which regulations are applicable to an academic research lab. Following that, a method to implement a QM-system will be presented. This method is shown as a project schedule and explains the requirements that are necessary to establish quality standards. The QM-system was implemented in the Laboratory for Molecular Biology and Functional Genomics of the Technical University of Applied Sciences Wildau and was included within the scope of the certification of the university in the audit. Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus-859 SN - 0949-8214 VL - 14 SP - 49 EP - 56 ER - TY - JOUR A1 - Wang, Chong A1 - Grohme, Markus A1 - Mali, Brahim A1 - Schill, Ralph O. A1 - Frohme, Marcus T1 - Towards Decrypting Cryptobiosis—Analyzing Anhydrobiosis in the Tardigrade Milnesium tardigradum Using Transcriptome Sequencing JF - PLoS ONE N2 - Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade Milnesium tardigradum. Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-6029 SN - 1932-6203 VL - 9 IS - 3 ER - TY - JOUR A1 - Tillich, Ulrich M. A1 - Wolter, Nick A1 - Schulze, Katja A1 - Kramer, Dan A1 - Brödel, Oliver A1 - Frohme, Marcus T1 - High-throughput cultivation and screening platform for unicellular phototrophs JF - BMC Microbiology N2 - In this work we describe and test high-throughput methods with the model organism Synechocystis sp. PCC6803. The required technical automation for these processes was achieved with a Tecan Freedom Evo 200 pipetting robot. The cultivation was performed in 2.2 ml deepwell microtiter plates within a cultivation chamber outfitted with programmable shaking conditions, variable illumination, variable temperature, and an adjustable CO2 atmosphere. Each microtiter-well within the chamber functions as a separate cultivation vessel with reproducible conditions. The automated measurement of various parameters such as growth, full absorption spectrum, chlorophyll concentration, MALDI-TOF-MS, as well as a novel vitality measurement protocol, have already been established and can be monitored during cultivation. Measurement of growth parameters can be used as inputs for the system to allow for periodic automatic dilutions and therefore a semi-continuous cultivation of hundreds of cultures in parallel. The system also allows the automatic generation of mid and long term backups of cultures to repeat experiments or to retrieve strains of interest. KW - cyanobacteria KW - synechocystis KW - HTS KW - high throughput KW - automated cultivation Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-6007 SN - 1471-2180 VL - 14 IS - 239 ER - TY - JOUR A1 - Tillich, Ulrich M. A1 - Lehmann, Sandra A1 - Schulze, Katja A1 - Dühring, Ulf A1 - Frohme, Marcus T1 - The Optimal Mutagen Dosage to Induce Point-Mutations in Synechocystis sp. PCC6803 and Its Application to Promote Temperature Tolerance JF - PLoS ONE N2 - Random mutagenesis is a useful tool to genetically modify organisms for various purposes, such as adaptation to cultivation conditions, the induction of tolerances, or increased yield of valuable substances. This is especially attractive for systems where it is not obvious which genes require modifications. Random mutagenesis has been extensively used to modify crop plants, but even with the renewed interest in microalgae and cyanobacteria for biofuel applications, there is relatively limited current research available on the application of random mutagenesis for these organisms, especially for cyanobacteria. In the presented work we characterized the lethality and rate of non-lethal point mutations for ultraviolet radiation and methyl methanesulphonate on the model cyanobacteria Synechocystis sp. PCC6803. Based on these results an optimal dosage of 10–50 J/m2 for UV and either 0.1 or 1 v% for MMS was determined. A Synechocystis wildtype culture was then mutagenized and selected for increased temperature tolerance in vivo. During the second round of mutagenesis the viability of the culture was monitored on a cell by cell level from the treatment of the cells up to the growth at an increased temperature. After four distinct rounds of treatment (two with each mutagen) the temperature tolerance of the strain was effectively raised by about 2°C. Coupled with an appropriate in vivo screening, the described methods should be applicable to induce a variety of desirable characteristics in various strains. Coupling random mutagenesis with high-throughput screening methods would additionally allow to select for important characteristics for biofuel production, which do not yield a higher fitness and can not be selected for in vivo, such as fatty acid concentration. In a combined approach with full genome sequencing random mutagenesis could be used to determine suitable target-genes for more focused methods. Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-6186 SN - 1932-6203 VL - 7 IS - 11 ER - TY - GEN A1 - Tillich, Ulrich M. A1 - Grüber, Stefanie A1 - Frohme, Marcus T1 - Etablierung von Hochdurchsatz-Kultivierungs- und -Screeningmethoden für phototrophe Einzeller T2 - Wissenschaftliche Beiträge 2012 N2 - Mit Hochdurchsatz-Kultivierungs- und -Screeningmethoden können viele Proben parallel, miniaturisiert und kostengünstig bearbeitet werden. Für phototrophe Organismen wie Mikroalgen und Cyanobakterien sind Hochdurchsatz-Kultivierungsverfahren jedoch bis heute kaum etabliert. Im Rahmen dieser Arbeit wurden diese Verfahren beispielhaft für das Cyanobakterium Synechocystis sp. PCC 6803 etabliert. Die benötigte technische Automatisierung wurde hierbei durch den Einsatz eines Tecan Genesis RSP 150 Pipettierroboters erreicht. Die Kultivierung erfolgte in Deepwell-Mikrotiterplatten innerhalb einer speziell angefertigten Kammer mit programmierbaren Schüttlern, einstellbarer Belichtung und CO2-Atmosphäre. Die in diesem System erreichten Wachstumsraten sind vergleichbar mit publizierten Kultivierungsmethoden. Das Hochdurchsatz-Screening wurde mit Hilfe eines in den Roboter integrierten Tecan Genios Plus Plattenreaders durchgeführt. Es wurden beispielhaft Methoden zur Bestimmung von optischer Dichte und Chlorophyllgehalt etabliert. Die hier vorgestellte Plattform kann vielseitig zur Analyse phototropher Organismen eingesetzt werden und ist durch entsprechende Assays leicht zur Messung anderer Parameter erweiterbar. N2 - High-throughput cultivation and screening methods allow a parallel, miniaturized and cost efficient processing of many samples. These methods however, have not been generally established for phototrophic organisms such as microalgae or cyanobacteria. In this work we describe and test high-throughput methods with the model organism Synechocystis sp. PC6803. The required technical automation for these processes was archived with a Tecan Genesis RSP 150 pipetting robot. The cultivation was performed in deepwell microtiter plates within a specially constructed cultivation chamber. The chamber is outfitted with programmable shaking conditions, variable illumination and an adjustable CO2 atmosphere. The growth rates archived within this system are comparable to those achieved with established methods such as bioreactors. The high-throughput screening was achieved with a Tecan Genios Plus plate reader integrated within the pipetting robot. Methods for determination of optical density and amount of chlorophyll were established within the scope of this work. The presented platform can be used for a variety of analyses of phototrophic organisms and is easily expandable with further assays to screen for additional targets. Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus-1335 SN - 0949-8214 VL - 16 SP - 31 EP - 36 ER - TY - THES A1 - Tillich, Ulrich M. T1 - Adaptive Evolution und Screening bei Cyanobakterien - Erzeugung und Untersuchung von Thermotoleranz in Synechocystis sp. PCC 6803 N2 - Ziel dieser Arbeit war die Erhöhung der Temperaturtoleranz des Cyanobakteriums Synechocystis sp. PCC 6803 mittels ungerichteter Mutagenese und adaptiver Evolution. Trotz des erneuten Interesses an Cyanobakterien und Mikroalgen in den letzten Jahren, gibt es nur relativ wenige aktuelle Studien zum Einsatz dieser Methoden an Cyanobakterien. Zur Analyse eines mittels Mutagenese erzeugten Gemischs an Stämmen, ist es von großem Vorteil Hochdurchsatz-Methoden zur Kultivierung und zum Screening einsetzen zu können. Auf Basis eines Pipettierroboters wurde solch eine Plattform für phototrophe Mikroorganismen neu entwickelt und folgend stetig verbessert. Die Kultivierung erfolgt in 2,2ml Deepwell-Mikrotiterplatten innerhalb einer speziell angefertigten Kultivierungskammer. Schüttelbedingungen, Beleuchtung, Temperatur und CO2-Atmosphäre sind hierbei vollständig einstellbar.Die Plattform erlaubt semi-kontinuierliche Kultivierungen mit automatisierten Verdünnungen von hunderten Kulturen gleichzeitig. Automatisierte Messungen des Wachstums, des Absorptionsspektrums, der Chlorophyllkonzentration, MALDI-TOF-MS sowie eines neu entwickelten Vitalitätsassays wurden etabliert. Für die Mutagenese wurden die Letalität- und die nicht-letale Punktmutationsrate von ultravioletter Strahlung und Methylmethansulfonat für Synechocystis charakterisiert. Synechocystis wurde mit den so ermittelten optimalen Dosen mehrfach behandelt und anschließend einer in vivo Selektion unterzogen. Somit wurde dessen Temperaturtoleranz um bis zu 3°C erhöht. Über die Screeningplattform wurden die thermotolerantesten monoklonalen Stämme identifiziert. Nach einer Validierung wurde das vollständige Genom der Stämme sequenziert. Hierdurch wurden erstmals Mutationen in verschiedenen Genen mit der Langzeittemperaturtoleranz von Synechocystis in Verbindung gebracht. Bei einigen dieser Gene ist es sehr unwahrscheinlich, dass sie mittels anderer Verfahren hätten identifiziert werden können. N2 - The goal of this work was the increase of the thermal tolerance of the cyanobacteria Synechocystis sp. PCC 6803 via random mutagenesis and adaptive evolution. Even with the renewed interest in cyanobacteria in the recent years, there is relatively limited current research available on the application of these methods on cyanobacteria. To analyse a mixture of various strains typically obtained through random mutagenesis, a method allowing high-throughput miniaturized cultivation and screening is of great advantage. Based on a pipetting robot a novel high-throughput screening system suitable for phototrophic microorganisms was developed and then constantly improved. The cultivation was performed in 2,2 ml deepwell microtiter plates within a cultivation chamber outfitted with programmable shaking conditions, variable illumination, variable temperature, and an adjustable CO2 atmosphere. The platform allows semi-continuous cultivation of hundreds of cultures in parallel. Automated measurements of growth, full absorption spectrum, chlorophyll concentration, MALDI-TOF-MS, as well as a novel vitality measurement protocol, have been established. Prior to the mutagenesis, the lethality and rate of non-lethal point mutations of ultraviolet radiation and methyl-methanesulphonate were characterized for Synechocystis. The thus determined optimal dosages were applied to Synechocystis followed by in vivo selection in four rounds of mutagenesis, thereby raising its temperature tolerance by 3°C. The screening platform was used to identify the most thermotolerant monoclonal strains. After validation, their whole genomes were sequenced. Thus mutations in various genes were identified which promote the strains'' thermal tolerance. For some of the genes it is very unlikely that their link to high thermal tolerance could have been identified by other approaches. KW - liquid handling KW - Cyanobakterien KW - Thermotoleranz KW - Adaptive Evolution Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-18307 ER - TY - JOUR A1 - Thiemann, Alexander A1 - Fu, Junjie A1 - Seifert, Felix A1 - Grant-Downton, Robert T A1 - Schrag, Tobias A A1 - Pospisil, Heike A1 - Frisch, Matthias A1 - Melchinger, Albrecht E A1 - Scholten, Stefan T1 - Genome-wide meta-analysis of maize heterosis reveals the potential role of additive gene expression at pericentromeric loci JF - BMC Plant Biology N2 - The identification of QTL involved in heterosis formation is one approach to unravel the not yet fully understood genetic basis of heterosis - the improved agronomic performance of hybrid F1 plants compared to their inbred parents. The identification of candidate genes underlying a QTL is important both for developing markers and determining the molecular genetic basis of a trait, but remains difficult owing to the large number of genes often contained within individual QTL. To address this problem in heterosis analysis, we applied a meta-analysis strategy for grain yield (GY) of Zea mays L. as example, incorporating QTL-, hybrid field-, and parental gene expression data. KW - heterosis KW - maize KW - QTL KW - grain yield KW - additive gene expression Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-5999 SN - 1471-2229 VL - 14 IS - 88 ER - TY - JOUR A1 - Tannenberg, Robert A1 - Tscheuschner, Georg A1 - Raab, Christopher A1 - Flemig, Sabine A1 - Döring, Sarah A1 - Ponader, Marco A1 - Thurmann, Melinda A1 - Paul, Martin A1 - Weller, Michael G. T1 - Simplified Sample Preparation and Lateral Flow Immunoassay for the Detection of Plant Viruses JF - Biosensors N2 - Lateral flow immunoassays (LFAs) are widely used for on-site testing; however, their use for the rapid detection of plant viruses in the field is often limited by inconvenient sample preparation. Here, we present a new sampling method and a simplified dipstick LFA format for the detection and monitoring of cowpea chlorotic mottle virus (CCMV) as a model plant pathogen. The assay employs a monoclonal mouse antibody for capture and a poly-clonal rabbit antibody conjugated to 80 nm gold nanoparticles for detection. Conventional sample and conjugate pads are omitted, allowing the test strips to be dipped directly into wells containing plant extract and antibody–gold conjugate. No plastic casing was required, which could lead to a reduction in waste. It was shown that CCMV concentrations as low as 3.5 μg/L or 350 pg per sample could be reliably detected in 15 min. Specificity tests confirmed that other plant viruses, cowpea mosaic virus (CPMV) and tobacco mosaic virus (TMV), did not produce false-positive results. In addition, we describe a new method for on-site sampling using a manual punch and a syringe equipped with a frit. This step combines grinding the sample, extraction, filtration, and reconstitution and mixing of the antibody-gold conjugate, enabling the analysis of punched leaf disks without laboratory equipment. When applied to CCMV-infected cowpea plants, the assay revealed systemic infection before visual symptoms became apparent. This work demonstrates that simplified LFAs combined with innovative sampling techniques can provide sensitive, specific, and rapid diagnostics for crop monitoring and support early intervention strategies in agriculture. KW - LFA format KW - Vigna unguiculata KW - cowpea chlorotic mottle virus KW - crop monitoring KW - lateral flow immunoassay KW - plant virus KW - sample preparation KW - waste reduction Y1 - 2026 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-21294 VL - 16 IS - 2 PB - MDPI ER -