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

这项工作是由来自马萨诸塞州波士顿（LSH）和R15 GM86805从NIH大学（LSH）一约瑟夫·P·希利资助。 SMP是部分地由赛诺菲 - 健赞奖学金波士顿马萨诸塞州大学的支持。

1.准备产孢注:在本协议中所描述的媒体使用标准配方和方法制成的13,17 表1给出了1升在本协议中所使用的各种媒体的表述。 <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;">细菌蛋白胨</td><td style="width: 50px;">酵母抽提物</td><td style="width: 50px;">细菌用琼脂</td><td style="width: 60px;">葡萄糖</td><td style="width: 70px;">醋酸钾</td><td style="width: 60px;">甘油</td><td style="width: 60px;"> DDH 2 O的</td></tr><tr height="15"><td height="15" style="height:20px;">耀皮玻璃板块</td><td> 20克</td><td> 10克</td><td> 20克</td><td> - </td><td> - </td><td> 30毫升</td><td> 970毫升</td></tr><tr height="15"><td height="15" style="height:20px;"> YPD平板</td><td> 20克</td><td> 10克</td><td> 20克</td><td> 20克</td><td> - </td><td> - </td><td>千毫升</td></tr><tr height="15"><td height="15" style="height:20px;"> YPD液体</td><td> 20克</td><td> 10克</td><td> - </td><td> 20克</td><td> - </td><td> - </td><td>千毫升</td></tr><tr height="15"><td height="15" style="height:20px;"> YPA液体</td><td> 20克</td><td> 10克</td><td> - </td><td> - </td><td> 20克</td><td> - </td><td>千毫升</td></tr><tr height="15"><td style="height: 20px; width: 80px;">产孢媒体</td><td> - </td><td> - </td><td> - </td><td> - </td><td> 10克</td><td> - </td><td>千毫升</td></tr></tbody></table> 表1:培养基配方用量给出了1升的介质。有关媒体的细节成份可以在材料表中找到。 <ol><li>如果所有的孢子细胞将是相同的基因型，步骤如下: <ol><li>解冻的酵母菌株被使用到YPG（YPGlycerol）板。在30℃下生长的酵母为12-16小时。重要的是，SK1细胞也准备形成孢子，所以SK1菌株不应留在YPG平板太长，或者它们可形成孢子在平板上。 注:生长在YPG新鲜解冻的菌株是优选的，如甘油作为碳源的利用需要氧化磷酸化，并由此选择为具有官能线粒体DNA的细胞。 SK1细胞有失去功能的线粒体DNA，并成为PETITES的倾向。需要对孢子形成功能性线粒体DNA。 </li><li>从YPG板到YPD（YPDextrose）板单菌落连胜酵母。在30℃生长在YPD板24-28小时酵母。 </li><li>大约36-48小时在YPD固体培养基中生长后，使用单个菌落开始25毫升的YPD培养从YPD板。直到细胞约有600 OD = 4.0〜7.0增长这种文化。在振荡培养箱中过夜进行，在30℃这一步骤中，以220rpm振荡。 </li><li>从YPD培养，使用细胞的适量启动千毫升YPA（YPAcetate）培养的即外径600 = 0.1的每个96孔板需要在第2节中30℃振荡培养箱中生长的酵母，在220（振荡转），直到文化OD 600为1.3〜1.5之间为。 注:接种100ml YPA文化的每个96多孔板第2节例如进行筛选，筛选如果96 4多孔板，将需要400毫升YPA文化。与YPA培养10％的额外体积开始是必要的，以防止蒸发体积损失。通常情况下，这将需要12-16小时之间，用于细胞达到相应的OD 600;的时间量可以根据酵母菌株的基因型变化。 YPA是presporulation媒体。 Pregrowth在醋酸有助于诱导的基因的表达（如IME1）所需的高效率和同步孢子形成6。 </li><li>旋在台式离心机1800×g离心5分钟以沉淀在YPA生长的细胞。洗涤细胞1倍与0.5体积的无菌高压灭菌去离子水。再次旋转，并丢弃洗。悬浮细胞在1×体积孢子形成介质。继续第2节。 </li></ol></li><li>如果使用对不同基因型的细胞孢子形成（ 即 ，具有不同的质粒转化的或含有不同的突变，典型地获得并冷冻储存存储），阵列单元的不同基因型到96多孔格式。然后，按照此步骤: <ol><li>使用无菌96尖头青蛙从解冻的冷冻库存96多孔板（S）转移到细胞耀皮玻璃固体介质。生长在30℃酵母过夜。 </li><li>使用无菌96尖头青蛙，从耀皮玻璃固体介质转移到细胞YPD固体培养基或选择性固体培养基（ 即 ，合成的基本培养基缺乏氨基酸，如果细胞中含有需要选择的质粒）。直到菌落形成每个菌株，通常为1一天YPD和2天，如果选择培养基是必要的，在30℃生长酵母。 </li><li>等分试样1.2毫升YPD液体培养基或选择性液体培养基中于96深2毫升多孔板的每个孔中。使用青蛙从固体介质为液体介质传输单元，覆盖由胶带固定就位的塑料盖，并在30℃下摇床生长过夜，以220rpm振荡。 注意:要尽量减少污染，使用塑料盖从矩形培养皿盖板。持用一块胶带代替盖，使空气循环最低限度地受到限制。 </li><li>在1800 XG 5分钟离心板，使用带适配器吊桶式离心机，以适应96孔板。倒掉从细胞介质;加入500μlYPA至每个孔，并重新悬浮沉淀的细胞在YPA。盖与由胶带固定就位的塑料盖。 </li><li>生长细胞15小时，在30℃下在振荡培养箱中，以220rpm振荡。继续第2节。 </li></ol></li></ol> 2.在96多孔形式孢子细胞<ol><li>添加任一种3毫米无菌固体玻璃珠或灭菌的5毫米×2毫米磁性搅拌棒放入一个96孔板的每个孔与1.3毫升井。 （见下文代表性的结果是否使用珠子或搅拌棒的讨论。） </li><li>如果要筛选的所有细胞都是一样的基因型，去2.2.1。如果细胞中筛选出不同基因型的，到2.2.2。 <ol><li>采取从步骤1.1.5和等分500μl的细胞加培养基的细胞进入96多孔板的每个孔中。与磁带就位塑料盖盖。继续执行2.3。 </li><li>自旋细胞是在YPA从1.2.4在1800×g离心5分钟，使用具有适配器吊桶式离心机，以容纳96孔板中。倒从细胞中˚F媒体。加入500微升孢子形成介质到每个孔中，并重新悬浮沉淀的细胞在孢子形成介质。与磁带就位塑料盖盖。继续执行2.3。 </li></ol></li><li>如果使用玻璃珠，在30℃的振荡培养箱地方样品和摇动以220rpm于孢子形成。如果使用磁力搅拌棒，将样品上的轰动板30℃培养箱孢子内。 注:用于磁性搅拌棒的确切搅拌速度不是很重要的，只要所有条大力移动和将培养物充气。 </li><li>通过孢子形成监测进展，从生成孢子的培养撤回样品进行可视化，从生成孢子的培养物添加6微升细胞对孢子形成介质中的24微升在96孔盖玻片板（1:5稀释）。离开细胞使用倒置显微镜检查细胞之前沉降5分钟。 注意:在野生型细胞SK1，紧凑成熟的孢子将形成由3转移到产孢媒体后6小时。通过孢子形成的进展可以通过显微镜使用荧光标记物，诸如核标记物来监测（ 即 ，HTB2-mCherry 18）来检查减数分裂或prospore膜标记物（ 即 ，G20 19），以检查孢子形态。或者，折射孢子的形成可使用诺马斯基DIC的显微镜开始转移到孢子形成介质后约12小时可以看到。 </li></ol>

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The authors have no competing financial interests.

在这里，我们提出了一个协议，用于在96多孔形式孢子SK1酵母。曝气是有效孢子形成，这需要使用任一搅拌棒或在每一个玻璃珠孔的关键。当细胞在96多孔板中形成孢子在没有任何一个珠或搅拌棒的振荡培养箱中，细胞不能有效地形成孢子。当细胞没有任何一个珠或在振荡培养箱搅拌棒相比，是在30℃下不搅拌（ 表1形成孢子仅在孢子形成效率的少量增加被看见;在摇对10％17％放置在搅?...

在芽殖酵母孢子形成进行了研究减数分裂1，基因重组2的机制，开发由细胞信号3的控制下，开发4的营养控制，转录期间提供见解生物学的许多方面，包括染色体分离的控制发展5调控，以及孢子形成6考试。孢子形成包括涉及的新的膜隔室的母细胞，随后用保护孢子壁6的沉积中形成的新的细胞分裂事件。这些研究，考察形成孢子的细胞经常采取迅速孢子酵母菌株SK1，其可以经受孢子形成的过程中约24小时以相对有效的方式7,8的优点。虽然对芽殖酵母产孢条件的优化已经被描述9-13，这些实验S于固体培养基或在孢子形成是利用培养管或瓶中大规模液体培养观察孢子形成。 在这里，我们描述了在96多井板格式形成孢子酵母的方法。我们发现，对于这种方法，曝气是用于同步和有效的孢子形成关键的，并且已经设计技术，以确保有足够的孢子形成的小体积多孔形式。在96多井板格式孢子允许使用高通量技术和多孔板格式，这种筛选使用平铺库14-16高拷贝抑制优化试剂进行筛选细胞。

<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>为了评估这个协议中，从在多孔板形成孢子的细胞（如上述）获得的孢子形成的效率进行了比较，在瓶（ 表2）使用较大体积的形成孢子的细胞。使用多孔板的没有达到在瓶中，在〜80％的效率可常规看出形成孢子时见到的高效率。在与适当的曝气（通过玻璃珠或搅拌棒提供的）多孔板形成孢子可以达到足够的孢子形成的效率大于50％，用一个5毫米×2mm的?...

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.

高效产孢<em&gt;酿酒酵母</em&gt;在96多孔形式

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

efficient-sporulation-saccharomyces-cerevisiae-96-multiwell

Research

JoVE Journal

Biology

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

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

视频: 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.

剖析酿酒酵母 ASCI

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

科研

生物学

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.

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

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测定确定出芽酵母中剪接体突变体的底物特异性敏感性

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

遗传学

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.

使用液体色谱加上串联质谱法的 糖核 酸菌的定量元细胞学

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

生物化学

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.

冷冻电子断层扫描 的糖核 酸的准备和冷冻-FIB微加工

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.

微阵列分析FOR酿酒酵母</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.

条形码酵母图书馆竞争力的基因屏幕

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.

流式细胞仪分离纯化<em&gt; S.酵母</em&gt;合子

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.

定向进化法<em&gt;酿酒酵母</em&gt;:突变体库的创建和筛选

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.

酿酒酵母间歇培养中的指数生长动力学分析呼吸和发酵代谢

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

高效产孢

摘要

探索更多视频

此视频中的章节

剖析酿酒酵母 ASCI

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

ACT1-CUP1测定确定出芽酵母中剪接体突变体的底物特异性敏感性

使用液体色谱加上串联质谱法的糖核酸菌的定量元细胞学

冷冻电子断层扫描的糖核酸的准备和冷冻-FIB微加工

微阵列分析FOR酿酒酵母

条形码酵母图书馆竞争力的基因屏幕

流式细胞仪分离纯化

定向进化法

酿酒酵母间歇培养中的指数生长动力学分析呼吸和发酵代谢

高效产孢

摘要

探索更多视频

此视频中的章节

剖析酿酒酵母 ASCI

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

ACT1-CUP1测定确定出芽酵母中剪接体突变体的底物特异性敏感性

使用液体色谱加上串联质谱法的 糖核 酸菌的定量元细胞学

冷冻电子断层扫描 的糖核 酸的准备和冷冻-FIB微加工

微阵列分析FOR酿酒酵母

条形码酵母图书馆竞争力的基因屏幕

流式细胞仪分离纯化

定向进化法

酿酒酵母间歇培养中的指数生长动力学分析呼吸和发酵代谢

使用液体色谱加上串联质谱法的糖核酸菌的定量元细胞学

冷冻电子断层扫描的糖核酸的准备和冷冻-FIB微加工