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 - Schulze, Katja A1 - Tillich, Ulrich M. A1 - Dandekar, Thomas A1 - Frohme, Marcus T1 - PlanktoVision - an automated analysis system for the identification of phytoplankton JF - BMC Bioinformatics N2 - Phytoplankton communities are often used as a marker for the determination of fresh water quality. The routine analysis, however, is very time consuming and expensive as it is carried out manually by trained personnel. The goal of this work is to develop a system for an automated analysis. Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-6364 SN - 1471-2105 VL - 14 IS - 115 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 - 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 -