Etablierung von Hochdurchsatz-Kultivierungs- und -Screeningmethoden für phototrophe Einzeller

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.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

Automatisierung des Bio-Plex Pro Analyseverfahrens

Für den simultanen Nachweis mehrerer Analyten innerhalb einer Probe ist die Bead-basierte Multiplexanalytik ein häufig verwendetes Verfahren und wird beispielsweise zur Quantifizierung von Proteinen genutzt. Die aufwendige Durchführung der Assays soll durch Automatisierung einerseits dem Anwender abgenommen werden, während andererseits die Genauigkeit und Reproduzierbarkeit der Analyse gesteigert wird. Die Automatisierung des Bio-Plex Pro™ Assays ist mit dem Pipettierroboter Tecan Freedom EVO 200 umgesetzt worden. Es ist ein Skript mit der Software Freedom EVOware® entwickelt worden, welches die Probenvorbereitung des Assays vollständig übernimmt. Für einen Vergleich der manuellen und automatisierten Methode sind die humanen Zytokine IL-2, IL-4, IL-10, GM-CSF, IFN-γ und TNF-α in einer achtstufigen Standardverdünnungsreihe und in unterschiedlich konzentrierten Proben gemessen worden. Die Berechnung der Streuungen (Standardabweichung und Variationskoeffizient) der einzelnen Standardverdünnungsreihen sowie der Vergleich von gemessenen und erwarteten Konzentrationen der automatisierten und manuellen Methode zeigen, dass die Automatisierung neben der zeitlichen Optimierung auch die Genauigkeit und Reproduzierbarkeit der Analyse verbessert.Bead-based multpiplex analysis is frequently used for the simultaneous detection of multiple analytes within a sample. Such assays are commonly used to quantify proteins. The automation of the process relieves the user from the complex assay conductance and on the other hand increases the accuracy and reproducibility of the analysis. The automation of the Bio-Plex Pro™ assay has been successfully implemented with the pipetting-robot Tecan Freedom EVO 200. A script has been developed, using the Freedom EVOware® which has the ability to perform the complete assay procedure. For a comparison between manual and automated methods, the human cytokines IL-2, IL-4, IL-10, GM-CSF, IFN-γ and TNF-α were analysed by an eight-point standard dilution series and with samples of different concentrations. The calculation of the variances (standard deviation and coefficient of variation) of the single standard dilution series as well as the comparison of the observed and expected concentrations of the manual or automated method show the higher precision and reproducibility of the automated process in addition to its time-saving nature

A simple viability analysis for unicellular cyanobacteria using a new autofluorescence assay, automated microscopy, and ImageJ

<p>Abstract</p> <p>Background</p> <p>Currently established methods to identify viable and non-viable cells of cyanobacteria are either time-consuming (eg. plating) or preparation-intensive (eg. fluorescent staining). In this paper we present a new and fast viability assay for unicellular cyanobacteria, which uses red chlorophyll fluorescence and an unspecific green autofluorescence for the differentiation of viable and non-viable cells without the need of sample preparation.</p> <p>Results</p> <p>The viability assay for unicellular cyanobacteria using red and green autofluorescence was established and validated for the model organism <it>Synechocystis </it>sp. PCC 6803. Both autofluorescence signals could be observed simultaneously allowing a direct classification of viable and non-viable cells. The results were confirmed by plating/colony count, absorption spectra and chlorophyll measurements. The use of an automated fluorescence microscope and a novel ImageJ based image analysis plugin allow a semi-automated analysis.</p> <p>Conclusions</p> <p>The new method simplifies the process of viability analysis and allows a quick and accurate analysis. Furthermore results indicate that a combination of the new assay with absorption spectra or chlorophyll concentration measurements allows the estimation of the vitality of cells.</p

PlanktoVision – an automated analysis system for the identification of phytoplankton

Background 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. Results A novel open source system for the automated recognition of phytoplankton by the use of microscopy and image analysis was developed. It integrates the segmentation of the organisms from the background, the calculation of a large range of features, and a neural network for the classification of imaged organisms into different groups of plankton taxa. The analysis of samples containing 10 different taxa showed an average recognition rate of 94.7% and an average error rate of 5.5%. The presented system has a flexible framework which easily allows expanding it to include additional taxa in the future. Conclusions The implemented automated microscopy and the new open source image analysis system - PlanktoVision - showed classification results that were comparable or better than existing systems and the exclusion of non-plankton particles could be greatly improved. The software package is published as free software and is available to anyone to help make the analysis of water quality more reproducible and cost effective

High-throughput cultivation and screening platform for unicellular phototrophs

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

The Optimal Mutagen Dosage to Induce Point-Mutations in <em>Synechocystis</em> sp. PCC6803 and Its Application to Promote Temperature Tolerance

<div><p>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 <em>Synechocystis</em> 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 <em>Synechocystis</em> wildtype culture was then mutagenized and selected for increased temperature tolerance <em>in vivo</em>. 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 <em>in vivo</em> 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 <em>in vivo</em>, 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.</p> </div

Summary of the conditions and results of each round of mutagenesis.

*<p>Note that in the 3rd round of mutagenesis there was a technical problem with the temperation, and cultures were also temperated during the night cycle, leading to many more cells dying than usual. A recovery at lower temperature was used to rescue surviving cells.</p

Characterization of UV.

<p>Survival rate (green; triangle) and point mutation rate (blue; square) plotted over the administered dosage of UV irradiation. The counted colonies for the survival rate and the point mutation rate were normalized by setting the control to 100%.</p