Name: efficient sporulation of saccharomyces cerevisiae in a 96 multiwell format
Uploaded: 2016-09-17T14:00:00.000+00:00
Duration: 8 min 54 s
Description: Watch this Scientific Journal Video about Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format at JoVE.com

Diese Arbeit wurde von einem Joseph P. Healey Zuschuss von der University of Massachusetts Boston (LSH) und R15 GM86805 vom NIH (LSH) unterstützt. SMP wird teilweise durch eine Sanofi-Genzyme Fellowship an der University of Massachusetts Boston unterstützt.

1. Vorbereitung für Sporulation . Anmerkung: Die Medien in diesem Protokoll beschrieben werden , hergestellt unter Verwendung Standardrezepturen und -verfahren 13,17 Tabelle 1 gibt die Formulierung für 1 L der verschiedenen Medien in diesem Protokoll verwendet. <table border="1" cellpadding="0" cellspacing="0" height="180" width="469"><colgroup><col /><col /><col span="3" /><col /><col span="2" /></colgroup><tbody><tr height="15"><td height="15" style="height:20px;"></td><td style="width: 55px;"> Bactopepton </td><td style="width: 50px;"> Hefeextrakt </td><td style="width: 50px;"> Bacto Agar </td><td style="width: 60px;"> Traubenzucker </td><td style="width: 70px;"> KALIUMACETAT </td><td style="width: 60px;"> Glycerol </td><td style="width: 60px;"> ddH 2 O </td></tr><tr height="15"><td height="15" style="height:20px;"> YPG-Platten </td><td> 20 g </td><td> 10 g </td><td> 20 g </td><td> - </td><td> - </td><td> 30 ml </td><td> 970 ml </td></tr><tr height="15"><td height="15" style="height:20px;"> YPD-Platten </td><td> 20 g </td><td> 10 g </td><td> 20 g </td><td> 20 g </td><td> - </td><td> - </td><td> 1,000 ml </td></tr><tr height="15"><td height="15" style="height:20px;"> YPD Flüssigkeit </td><td> 20 g </td><td> 10 g </td><td> - </td><td> 20 g </td><td> - </td><td> - </td><td> 1,000 ml </td></tr><tr height="15"><td height="15" style="height:20px;"> YPA Flüssigkeit </td><td> 20 g </td><td> 10 g </td><td> - </td><td> - </td><td> 20 g </td><td> - </td><td> 1,000 ml </td></tr><tr height="15"><td style="height: 20px; width: 80px;"> Sporulation Medien </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 10 g </td><td> - </td><td> 1,000 ml </td></tr></tbody></table> Tabelle 1:. Medienformulierungen Die Beträge werden für 1 l Medien gegeben. Einzelheiten über MedienZutaten können in der Materialtabelle. <ol><li> Wenn alle sporulierendem Zellen die gleichen Genotyps sein, gehen Sie folgendermaßen vor: <ol><li> Thaw Hefestamm auf einem YPG (YPGlycerol) Platte verwendet werden. Wachsen Hefe für 12-16 h bei 30 ° C. Wichtig ist, dass SK1 Zellen auch sollte, so SK1 Stämme zur Sporulation balanciert nicht zu lange auf YPG-Platten belassen werden, oder sie können auf der Platte sporulieren. Hinweis: Ein frisch aufgetauten Belastung auf YPG gezüchtet wird bevorzugt, wie Verwendung von Glycerin als Kohlenstoffquelle oxidative Phosphorylierung benötigt, und wählt somit für Zellen, die funktionelle mitochondrialen DNA aufweisen. SK1 Zellen haben eine Tendenz, funktionelle Mitochondrien-DNA zu verlieren und petites. Funktionelle mitochondrialer DNA ist für die Sporenbildung benötigt. </li><li> Streak Hefe aus der YPG Platte auf eine YPD (YPDextrose) Platte für einzelne Kolonien. Wachsen Hefe auf YPD-Platte 24-28 h bei 30 ° C. </li><li> Nach etwa 36 bis 48 h Wachstum auf YPD festen Medien, starten Sie eine 25 ml YPD-Kultur eine einzige Kolonie mitvon der YPD Platte. Wachsen diese Kultur , bis die Zellen etwa OD 600 = 4,0 bis 7,0 liegen. Führen Sie diesen Schritt bei 30 ° C über Nacht in einem Schüttelinkubator bei 220 rpm schütteln. </li><li> Von der YPD - Kultur, eine entsprechende Menge an Zellen verwenden , um 1000 ml YPA (YPAcetate) Kultur beginnen , die OD 600 = 0,1 für jede 96 - Well - Platte Bedarf in Abschnitt 2. wachsen Hefe in einem 30 ° C Schüttelinkubator ist (bei ​​220 Schütteln rpm) , bis die Kultur OD 600 zwischen 1,3 bis 1,5 ist. Hinweis: Impfen 100 ml YPA Kultur für jede 96 Multiwellplatte in Abschnitt 2. Zum Beispiel zu sehen sein, wenn vier 96 Mikrotiterplatten Screening, 400 ml YPA Kultur benötigt. Beginnend mit 10% mehr Volumen in der YPA Kultur ist notwendig, gegen Verdunstungsvolumenverlust zu schützen. Typischerweise wird es zwischen 12 bis 16 Stunden dauern , Zellen , die die entsprechenden OD 600 zu erreichen; die Zeit kann in Abhängigkeit vom Genotyp des Hefestamm variieren. YPA ist die presporulation Medien. Pregrowthin Acetat hilft Expression von Genen (wie IME1) induzieren für einen effizienten und synchronen Sporulation 6 benötigt. </li><li> Spin bei 1.800 xg in einer Tischzentrifuge für 5 min Zellen in YPA gewachsen zu pelletieren. Waschen Sie die Zellen 1x mit 0,5 Volumen sterilen autoklaviert entsalztem Wasser. Spin wieder, und entsorgen Sie waschen. Die Zellen in 1x Volumen Sporulation Medien. Weiter zu Abschnitt 2. </li></ol></li><li> Bei Verwendung von Zellen für die Sporulation , die von verschiedenen Genotypen sind, (dh transformiert mit verschiedenen Plasmiden oder verschiedene Mutationen enthalten, in der Regel erhalten , und als gefrorene Vorräte gespeichert), array Zellen der verschiedenen Genotypen in eine 96 - Multiwell - Format. Dann gehen Sie folgendermaßen vor: <ol><li> Verwenden Sie eine sterile 96 Zinke Frogger Zellen aus aufgetauten gefrorenen Bestände in 96 Multiwellplatte zu übertragen (s) auf YPG festen Medien. Wachsen Hefe über Nacht bei 30 ° C. </li><li> Mit einem sterilen 96 Zinke Frogger, Transfer-Zellen von YPG festen Medien auf YPD feste Medien oder selektivefeste Medien (dh synthetische Minimalmedien Aminosäuren fehlen, wenn die Zellen Plasmide enthalten , die Auswahl benötigen). Hefe wachsen bei 30 ° C bis Kolonien für jeden Stamm gebildet werden, typischerweise 1 Tag auf YPD und 2 Tage, wenn selektiven Medien notwendig. </li><li> Aliquot 1,2 ml YPD flüssigen Medien oder selektive flüssige Medien in jede Vertiefung einer 96 tief 2 ml Multiwellplatte. Transfer-Zellen aus festen Medien in flüssige Medien ein Frogger mit, Deckel mit einem Plastikdeckel an Ort und Stelle durch ein Band gehalten und wachsen über Nacht in 30 ° C Schüttler bei 220 Umdrehungen pro Minute schütteln. Hinweis: Zur Minimierung von Verschmutzung, Abdeckplatten mit den Kunststoffdeckel aus einem rechteckigen Petrischale. Halten Deckel anstelle ein Stück Klebeband, so dass die Luftzirkulation minimal eingeschränkt ist. </li><li> Zentrifugenplatten bei 1.800 × g für 5 min, eine Schwingbecherzentrifuge mit Adapter mit 96-Well-Platten aufzunehmen. Gießen Sie Medien von Zellen; Add 500 ul YPA in jede Vertiefung und resuspendieren in der YPA pelletierten Zellen. Abdeckungmit Kunststoffdeckel an Ort und Stelle durch ein Band gehalten. </li><li> Wachsen Zellen für 15 Stunden bei 30 ° C in einem Schüttelinkubator bei 220 rpm schütteln. Weiter zu Abschnitt 2. </li></ol></li></ol> 2. sporulierendem Zellen in einer 96-Multiwell-Format <ol><li> Fügen Sie entweder ein 3 mm sterilen festen Glasperle oder eine sterile 5 mm x 2 mm magnetischen Rührstab in jede Vertiefung einer 96-Well-Platte mit 1,3 ml Brunnen. (Siehe Repräsentative Ergebnisse unten für eine Diskussion darüber , ob eine Perle oder einen Rührstab zu verwenden.) </li><li> Wenn alle Zellen des gleichen Genotyps gescreent werden, um 2.2.1 gehen. Wenn Zellen von verschiedenen Genotypen gescreent werden, um 2.2.2 gehen. <ol><li> Nehmen Sie Zellen aus Schritt 1.1.5 und aliquoten 500 ul Zellen plus Medien in jede Vertiefung der 96 Multi-Well-Platte. Abdeckung mit Plastikdeckel an Ort und Stelle durch ein Band gehalten. Gehen Sie zu 2.3. </li><li> Spin-Zellen, die für 5 min in YPA von 1.2.4 bei 1.800 xg sind, eine Schwingbecherzentrifuge mit Adapter mit 96-Well-Platten aufzunehmen. Gießen vonf Medien aus Zellen. Hinzufügen 500 ul Sporulation Medium zu jeder Vertiefung gegeben und resuspendieren pelletierten Zellen in der Sporulation Medien. Abdeckung mit Plastikdeckel an Ort und Stelle durch ein Band gehalten. Gehen Sie zu 2.3. </li></ol></li><li> Bei Verwendung einer Glasperle, Platz Proben in einem Schüttelinkubator bei 30 ° C und Schütteln bei 220 Umdrehungen pro Minute für die Sporenbildung. Wenn eine magnetische Rührstab verwenden, Ort Proben auf einer Rührplatte innerhalb einer 30 ° C-Inkubator für Sporulation. Hinweis: Die genaue Rührgeschwindigkeit für die magnetischen Rührstäbe nicht sehr wichtig ist, solange alle Balken bewegen sich energisch und die Kultur belüftet wird. </li><li> Zu überwachen, die Progression durch Sporulation werden Proben aus der Kultur sporulierenden zur Visualisierung, In 6 ul von Zellen aus der sporulierenden Kulturen auf 24 &amp; mgr; l der Sporulation Medium in einer 96-Well-Deckplatte (1: 5-Verdünnung). Lassen Sie Zellen für 5 Minuten absetzen, bevor Zellen mit einem inversen Mikroskop zu untersuchen. Hinweis: In Wildtyp-SK1 Zellen, kompakt reifen Sporen von 3 bilden6 Stunden nach der Übertragung auf die Sporulation Medien. Progression durch Sporulation kann durch Mikroskopie unter Verwendung von fluoreszierenden Markern, wie beispielsweise ein Kernmarker (dh HTB2-mCherry 18) zu untersuchen , die meiotische Teilung oder eine prospore Membranmarker (dh G20 19) zu untersuchen sporen Morphogenese überwacht werden. Alternativ kann die Bildung von refractile Sporen Nomarski DIC-Mikroskopie beginnend etwa 12 Stunden nach dem Transfer auf die Sporulation Medien gesehen werden. </li></ol>

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The authors have no competing financial interests.

Hier präsentieren wir ein Protokoll für sporulierendem SK1 Hefe in einem 96 Multi-Well-Format. Belüftung ist der Schlüssel für die effiziente Sporulation, die die Verwendung von entweder einem Rührstab oder einer Glasperle in jeder Vertiefung erfordert. Wenn Zellen ohne entweder in einer 96-Mikrotiterplatte in einem Schüttelinkubator sporulierten sind eine Perle oder einem Rührstab, Sporulation nicht effizient Zellen. Nur ein geringer Anstieg der Sporulationseffizienz gesehen wird , wenn Zellen ohne entweder ein...

Sporulation in der Bäckerhefe wurde untersucht , Einblicke in viele Aspekte der Biologie zu schaffen, einschließlich der Kontrolle der Chromosomensegregation während der Meiose 1, Mechanismen der genetischen Rekombination 2, die Kontrolle der Entwicklung von Zelle 3 Signalisierung, Ernährungs - Management von Entwicklung 4, die Transkriptions Regulierung der Entwicklung 5 und die Prüfung der Sporenbildung 6. Sporenbildung beinhaltet eine neuartige Zellteilung Ereignis die Bildung neuer Membran Kompartimente innerhalb der Mutterzelle durch Abscheiden einer Schutzsporenwand 6 gefolgt beteiligt sind . Diese Studien , die sporulierendem Zellen oft nutzen die schnell sporulierendem Hefestamm SK1 zu untersuchen, die den Prozess der Sporulation in etwa 24 Stunden in einem relativ effiziente Art und Weise 7,8 eingehen können. Obwohl die Optimierung der Sporulationsbedingungen für Hefe - Knospung haben 9-13 beschrieben worden ist , diese Experiments geprüft Sporulation auf festen Medien oder in größerem Maßstab flüssige Kulturen, in denen die Sporenbildung durchgeführt wird Kulturröhrchen oder Kolben verwendet wird. Hier beschreiben wir ein Verfahren zur sporulierenden Hefe in einem 96-Multiwell-Platten-Format. Wir finden, dass für dieses Verfahren, Belüftung für synchrone und effiziente Sporulation kritisch ist, und haben Techniken entwickelt, um eine ausreichende Sporulation ein kleinvolumiger Multiwell-Format zu gewährleisten. In einer 96 Multi - Well - Plattenformat sporulierendem ermöglicht Zellen für eine Multi - Well - Plattenformat, wie zum Screening für eine hohe Kopien Suppressoren mit einem gekachelten Bibliothek 14-16 optimierte Hochdurchsatztechniken und unter Verwendung von Reagenzien zu sehen sein.

<table><tbody><tr><td>Nunc 1.3 ml DeepWell Plates</td><td>ThermoScientific</td><td>260251</td><td>Used for sporulation</td></tr><tr><td>Nunc 2.0 ml DeepWell plates</td><td>ThermoScientific</td><td>278743</td><td>Used for presporulation growth, step 1.2.3</td></tr><tr><td>3 mm glass bead</td><td>Fisher</td><td>11-312A</td><td>Used for sporulation</td></tr><tr><td>5 mm x 2 mm stir bar, pack of 12</td><td>Fisher</td><td>14-511-82</td><td>Used for sporulation</td></tr><tr><td>96 well frogger</td><td>V&amp;amp;P Scientific</td><td>VP407</td><td>needed for step 1.2</td></tr><tr><td>library copier</td><td>V&amp;amp;P Scientific</td><td>VP381</td><td>needed for step 1.2; to be used with the frogger</td></tr><tr><td>rectangular Petri dish</td><td>ThermoScientific</td><td>264728</td><td>needed for step 1.2</td></tr><tr><td>Bacto Peptone</td><td>BD</td><td>211677</td><td>needed for media</td></tr><tr><td>Yeast Extract</td><td>BD</td><td>212750</td><td>needed for media</td></tr><tr><td>Bacto Agar</td><td>BD</td><td>212750</td><td>needed for media</td></tr><tr><td>Dextrose</td><td>Fisher</td><td>D16-3</td><td>needed for media</td></tr><tr><td>Potassium Acetate</td><td>Fisher</td><td>P171-500</td><td>needed for media</td></tr><tr><td>Glycerol</td><td>Fisher</td><td>G33-500</td><td>needed for media</td></tr><tr><td>Black 96 well glass bottom plate</td><td>MatTek</td><td>PBK96G-1.5.5-F</td><td>needed for step 2.4</td></tr></tbody></table>

efficient sporulation of saccharomyces cerevisiae in a 96 multiwell format

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.

<html> Um dieses Protokoll Beurteilung Sporulation Effizienzen aus sporulierenden Zellen in Multi - Well - Platten erhalten (wie oben beschrieben) wurden im Vergleich zu Zellen sporuliert Verwendung größerer Volumen in den Flaschen (Tabelle 2). Die Verwendung von Multiwell-Platten nicht erreicht die hohe Effizienz gesehen, wenn in Flaschen sporulierenden, wobei ~ 80% Wirkungsgrad routinemäßig gesehen werden kann. Sporulierendem in Mikrotiterplatten mit der richtigen B...

Watch this Scientific Journal Video about Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format at JoVE.com

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

Effiziente Sporulation von<em&gt; Saccharomyces cerevisiae</em&gt; In einer 96-Multiwell-Format

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for ...

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Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

10.1K Aufrufe.  University of Massachusetts Boston. Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

Serie: Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Präparation Saccharomyces cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Forschung

Biologie

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Ein High-Throughput-Verfahren zur global Studie der Organellen-Morphologie in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. Whether disease-related or functionally selected, many of these mutations have been studied using growth assays in the model organism Saccharomyces cerevisiae (yeast). The splicing-specific copper growth assay, or ACT1-CUP1 assay, provides a comprehensive analysis of mutation at the phenotypic level. The ACT1-CUP1 assay utilizes reporters that confer copper tolerance when correctly spliced. Thus, in the presence of copper, changes in yeast viability correlate to changes in mRNA production through splicing. In a typical experiment, the yeast spliceosome is challenged with different non-consensus splicing reporters and the splicing factor mutation of interest to detect any synergetic or antithetical impact on splicing. Here a full description of copper plate preparation, plating of yeast cells, and data evaluation are given. A selection of complimentary experiments is described, highlighting the versatility of the ACT1-CUP1 reporters. The ACT1-CUP1 assay is a handy tool in the splicing toolbox thanks to the direct read-out of mutational effect(s) and the comparative possibilities from the continuing use in the field.

The ACT1-CUP1 assay, a copper growth assay, provides a quick readout of precursor messenger RNA (pre-mRNA) splicing and the impact mutant splicing factors have on spliceosomal function. This study provides a protocol and highlights the customization possible to address the splicing question of interest.

ACT1-CUP1-Assays bestimmen die substratspezifischen Sensitivitäten von spleißeosomalen Mutanten in knospender Hefe

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. ...

Genetik

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

Quantitative Metabolomik von Saccharomyces Cerevisiae mittels Flüssigkeitschromatographie gekoppelt mit Tandem-Massenspektrometrie

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Biochemie

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

Herstellung und Kryo-FIB-Mikrobearbeitung von Saccharomyces cerevisiae für die Kryo-Elektronentomographie

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

Microarray-Analyse für Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Competitive Genomic Screens von Barcoded Hefe Bibliotheken

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

Durchflusszytometrie-basierte Reinigung von<em&gt; S. cerevisiae</em&gt; Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Entwicklung Methode Regie in<em&gt; Saccharomyces cerevisiae</em&gt;: Mutant Bibliothek Schöpfung und Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The fermentable carbon concentration mainly governs how the yeast cells generate ATP; thus, the variation in fermentable carbohydrate levels drives the energetic metabolism of S. cerevisiae. This paper describes a high-throughput method based on exponential yeast growth to estimate the effects of concentration changes and nature of the carbon source on respiratory and fermentative metabolism. The growth of S. cerevisiae is measured in a microplate or shaken conical flask by determining the optical density (OD) at 600 nm. Then, a growth curve is built by plotting OD versus time, which allows identification and selection of the exponential phase, and is fitted with the exponential growth equation to obtain kinetic parameters. Low specific growth rates with higher doubling times generally represent a respiratory growth. Conversely, higher specific growth rates with lower doubling times indicate fermentative growth. Threshold values of doubling time and specific growth rate are estimated using well-known respiratory or fermentative conditions, such as non-fermentable carbon sources or higher concentrations of fermentable sugars. This is obtained for each specific strain. Finally, the calculated kinetic parameters are compared with the threshold values to establish whether the yeast shows fermentative and/or respiratory growth. The advantage of this method is its relative simplicity for understanding the effects of a substance/compound on fermentative or respiratory metabolism. It is important to highlight that growth is an intricate and complex biological process; therefore, preliminary data from this method must be corroborated by the quantification of oxygen consumption and accumulation of fermentation byproducts. Thereby, this technique can be used as a preliminary screening of compounds/substances that may disturb or enhance fermentative or respiratory metabolism.

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Here we present a protocol to estimate the respiratory and fermentative metabolism by fitting the exponential growth of Saccharomyces cerevisiae to the exponential growth equation. Calculation of the kinetic parameters allows for the screening of influences of substances/compounds on fermentation or mitochondrial respiration.

Saccharomyces cerevisiae Exponentielles Wachstum Kinetik in Batch-Kultur, Atem- und fermentativen Stoffwechsel analysieren

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The ...

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