이 작품은 NIH에서 매사추세츠 보스턴 (LSH) 및 R15 GM86805 대학 (LSH)에서 조셉 P. 힐리 보조금에 의해 지원되었다. SMP는 매사추세츠 보스턴 대학에서 사노피 - 젠 자임 원정대에 의해 부분적으로 지원됩니다.

1. 포자 형성을위한 준비 참고 :.이 프로토콜에 설명 된 미디어는 표준 조리법과 방법을 사용하여 제조하는 13, 17 표 1은이 프로토콜에서 사용되는 다양한 미디어의 1 L에 대한 제제를 제공합니다. <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;"> 박토 펩톤 </td><td style="width: 50px;"> 효모 추출물 </td><td style="width: 50px;"> 박토 한천 </td><td style="width: 60px;"> 우선 당 </td><td style="width: 70px;"> 칼륨 아세테이트 </td><td style="width: 60px;"> 글리세린 </td><td style="width: 60px;"> DDH 2 O </td></tr><tr height="15"><td height="15" style="height:20px;"> YPG 판 </td><td> 20g </td><td> 10g </td><td> 20g </td><td> - </td><td> - </td><td> 30 ml의 </td><td> 970 ml의 </td></tr><tr height="15"><td height="15" style="height:20px;"> YPD 판 </td><td> 20g </td><td> 10g </td><td> 20g </td><td> 20g </td><td> - </td><td> - </td><td> 1,000 ml의 </td></tr><tr height="15"><td height="15" style="height:20px;"> YPD 액체 </td><td> 20g </td><td> 10g </td><td> - </td><td> 20g </td><td> - </td><td> - </td><td> 1,000 ml의 </td></tr><tr height="15"><td height="15" style="height:20px;"> YPA 액체 </td><td> 20g </td><td> 10g </td><td> - </td><td> - </td><td> 20g </td><td> - </td><td> 1,000 ml의 </td></tr><tr height="15"><td style="height: 20px; width: 80px;"> 포자 형성 미디어 </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 10g </td><td> - </td><td> 1,000 ml의 </td></tr></tbody></table> 표 1. 미디어 제제 금액은 미디어의 1 L에 대해 제공됩니다. 미디어에 대한 세부 사항재료는 재료 표에서 찾을 수 있습니다. <ol><li> 모든 포자 세포는 동일한 유전자형을 할 경우,이 절차를 따르십시오 : <ol><li> 해동 효모 균주는 YPG (YPGlycerol) 접시에 사용합니다. 30 ° C에서 12 ~ 16 시간 동안 효모 성장. 중요한 것은, SK1 세포도 그렇게 SK1 균주 YPG 판에 너무 오래 남아해서는 안 sporulate 태세, 또는 그들이 접시에 sporulate 수 있습니다. 참고 YPG 성장 갓 해동 균주가 탄소원으로서 글리세롤의 이용으로 산화 적 인산화 필요 바람직하고, 따라서 기능적 미토콘드리아 DNA가 세포에 대해 선택된다. SK1 세포 기능 미토콘드리아 DNA를 잃고 PETITES가되는 경향이있다. 기능 미토콘드리아 DNA는 포자 형성에 필요합니다. </li><li> 하나의 식민지에 대한 YPD (YPDextrose) 판 상에 YPG 판에서 행진 효모. 30 ° C에서 YPD 판 24 ~ 28 시간에 효모를 성장. </li><li> YPD 고체 배지에서 성장의 약 36-48 시간 후, 하나의 식민지를 사용하여 25 ml의 YPD 문화를 시작YPD 판에서. 세포는 7.0 = 4.0 OD (600)에 대해 때까지이 문화를 성장. 220 rpm으로 진탕, 진탕 배양기에서 하룻밤 30 ° C에서이 단계를 수행합니다. </li><li> YPD 문화에서 (220 진탕 30 ° C 진탕 배양기에서 효모 성장 2 제 각 96 웰 플레이트의 필요성에 대해 OD 600 = 0.1입니다 YPA (YPAcetate) 문화의 1,000 mL로 시작하는 세포의 적절한 양을 사용 OD (600)는 1.5-1.3 사이 인 문화까지 회전). 참고 : 네 96 멀티 웰 플레이트를 선별​​ 경우 각각 96 멀티 웰 플레이트에 대한 YPA 문화의 100 ㎖를 접종은, 예를 들어 제 2 절에서 상영 될, YPA 문화의 400 ml의 필요합니다. YPA 문화에서 10 % 추가 볼륨부터는 증발 볼륨 손실을 방지 할 필요가있다. 세포가 적절한 OD 600에 도달하기 위해 일반적으로는 12 ~ 16 시간 사이에 걸립니다; 시간은 효모 균주의 유전형에 따라 달라질 수있다. YPA는 presporulation 미디어입니다. Pregrowth아세테이트 유전자의 발현 (예 : IME1)을 효율적이고 동기 포자 6 필요를 유도하는 데 도움이됩니다. </li><li> 5 분을위한 테이블 탑 원심 분리기에서 1,800 XG에 스핀 YPA에서 자란 세포 펠렛합니다. 세척 세포는 멸균 멸균 탈 이온수의 0.5 볼륨 1 배. 다시 스핀, 씻어 버린다. 1 배 볼륨 포자의 미디어를 Resuspend 세포. 제 2 절을 계속합니다. </li></ol></li><li> 다른 유전자형,의이다 포자에 대한 세포를 사용하여 (다른 플라스미드로 형질 전환 즉, 또는 다른 일반적으로 획득 및 냉동 주식으로 저장 돌연변이 포함) 96 멀티 웰 형식으로 다른 유전자형의 배열 세포를합니다. 그런 다음이 절차를 따르십시오 : <ol><li> YPG 고체 배지에 96 멀티 웰 플레이트 (들)에 해동 냉동 주식에서 세포를 전송하는 멸균 96 구 프로 거를 사용합니다. 30 ° C에서 효모 밤새 성장. </li><li> 멸균 96 구 프로 거를 사용하여 YPD 고체 배지 또는 선택에 YPG 고체 배지에서 세포를 전송고체 미디어 (세포가 선택이 필요 플라스미드가 포함 된 경우 즉, 합성 최소 배지는 아미노산을 결여). 선택적 미디어가 필요한 경우 식민지가 이일 YPD에 일반적으로 1 일, 각 변형에 대해 형성 될 때까지 30 ° C에서 효모를 성장. </li><li> 나누어지는 96 깊은 2 ml의 멀티 웰 플레이트의 각 웰에 YPD 액체 매체 또는 선택적 액체 매체의 1.2 ml의. 프로 거를 사용하여 액체 매질에 고체 미디어에서 전송 세포는 테이프로 고정 플라스틱 뚜껑으로 덮고, 220 rpm으로 떨고, 30 ° C 통에 밤새 성장. 주 : 오염을 최소화하기 위해, 직사각형 페트리 접시에서 플라스틱 뚜껑을 사용하여 커버 플레이트. 공기 순환이 최소한으로 제한되어 있으므로 테이프의 조각을 사용하여 장소에 뚜껑을 잡으십시오. </li><li> 어댑터 스윙 버킷 원심 분리기를 이용하여 5 분 동안 1,800 XG에 원심 분리기 판은, 96 웰 플레이트를 수용한다. 세포에서 미디어를 붓고; YPA에서 펠렛 세포를 각각 500 μL의 YPA을 잘 추가하고 재현 탁. 덮개테이프로 고정 플라스틱 뚜껑. </li><li> 220 rpm으로 진탕, 진탕 배양기에서 30 ° C에서 15 시간 동안 세포를 성장. 제 2 절을 계속합니다. </li></ol></li></ol> 96 멀티 웰 형식 2. 포자 세포 <ol><li> 하나 3mm 멸균 고체 유리 구슬 또는 1.3 ml의 우물과 96 웰 플레이트의 각 웰에 멸균 5mm X 2mm 자기 교반 막대를 추가합니다. (비드 또는 교반 막대 사용 여부에 대한 설명은 아래 대표 결과를 참조하십시오.) </li><li> 스크리닝 될 수있는 모든 셀이 동일한 유전자형의 경우 2.2.1 간다. 상영하는 세포가 다른 유전자형의 경우, 2.2.2로 이동합니다. <ol><li> 96 멀티 웰 플레이트의 각 웰에 단계 1.1.5 나누어지는 세포 플러스 미디어 500 μl를에서 세포를 가져 가라. 테이프로 고정 플라스틱 뚜껑 커버. 2.3로 넘어갑니다. </li><li> 96 웰 플레이트를 수용하기 위해 어댑터 스윙 버킷 원심 분리기를 사용하여 1.2.4에서 5 분 1,800 XG에 YPA에 스핀 세포. 의 부어셀로부터 F 매체. 포자 미디어에서 펠렛 세포를 각 포자 매체의 500 μl를 잘 추가하고 재현 탁. 테이프로 고정 플라스틱 뚜껑 커버. 2.3로 넘어갑니다. </li></ol></li><li> 유리 비드를 사용하는 경우, 장소 30 ° C에서 진탕 배양기에서 샘플 및 포자 220 rpm으로 흔들. 포자 형성을위한 30 ° C 배양기의 내부에 교반 접시에 자기 교반 막대, 장소 샘플을 사용하는 경우. 참고 : 모든 바 정력적으로 이동하고 문화 폭기 된 바와 같이 자기 ​​교반 막대에 대한 정확한 교반 속도만큼 매우 중요하지 않습니다. </li><li> , 포자 형성을 통해 진행을 모니터링 시각화의 포자 배양에서 샘플을 철회, 96 웰 커버 슬립 판에 포자 미디어의 24 μL에 포자 배양에서 세포의 6 μl를 추가하는 방법 (1 : 5 희석). 거꾸로 현미경을 사용하여 세포를 검사하기 전에 5 분 동안 정착 세포를 남겨주세요. 주 : 야생형 SK1 세포 컴팩트 성숙한 포자 (3)에 의해 형성 할포자 매체로 전사 후 6 시간이다. 포자 형성을 통해 진행이 같은 핵 마커 형광 마커를 사용하여 현미경으로 모니터링 할 수 있습니다 (즉, Htb2-mCherry 18)가 감수 분열 또는 prospore 막 마커를 조사하는 (즉, G20 19) 포자 형태 형성을 검사합니다. 대안 적으로, 굴절 포자의 포자 형성을 용지에 전사 한 후 약 12​​ 시간을 시작 노멀 스키 DIC 현미경을 사용하여 알 수있다. </li></ol>

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The authors have no competing financial interests.

여기에서 우리는 96 멀티 웰 형식으로 SK1 효모를 포자를위한 프로토콜을 제시한다. 폭기는 교반 막대 또는 아니라 각각의 유리 구슬 중 하나의 사용을 필요로 효율적인 포자 형성을위한 열쇠입니다. 세포가 비드 또는 교반 막대 하나없이 진탕 배양기에서 96 멀티 웰 플레이트에 포자 경우, 세포가 효율적으로 sporulate하지 않습니다. 세포가 비드 또는 (교반없이 30 ° C에서 표 1 인에 비해...

신진 효모의 포자 형성은 감수 분열 (1), 유전자 재조합 2의 메커니즘 (3) 세포 신호에 의해 발전의 제어, 개발 4의 영양 관리, 전사시 염색체 분리의 제어를 포함 생물학의 여러 측면, 통찰력을 제공하기 위해 연구되고있다 개발 5의 규제 및 포자 형성 (6)의 시험. 포자 형성 보호 포자 벽 (6)의 증착에 이어 머더 셀 내의 새로운 멤브레인 구획의 형성을 포함하는 신규 한 세포 분열 이벤트를 포함한다. 포자 세포 검사 이러한 연구들은 상대적으로 효율적인 방식으로 7,8- 약 24 시간에 포자 형성 과정을 거칠 수있는 급속 포자 효모 SK1, 활용. 효모 신진 포자 조건의 최적화는 9-13,이 실험 설명되었지만S는 고체 배지 또는 포자를 배양 튜브 또는 플라스크를 사용하여 수행되는 대규모의 액체 배양 물에서 포자를 조사 하였다. 여기에서 우리는 96 멀티 웰 플레이트 형식으로 효모 포자하는 방법을 설명합니다. 우리는이 방법에 대해, 통기 동기와 효율적인 포자 형성에 중요한 것을 발견하고, 작은 볼륨 멀티 웰 형식으로 충분한 포자 형성을 보장하기 위해 기술을 고안했다. 세포는 높은 처리량 기술과 바둑판 라이브러리 14-16을 사용하여 높은 카피 억제하는 멀티 웰 플레이트 형식 그러한 스크리닝에 최적화 된 시약을 사용하여 스크리닝 될 수 있도록 96- 멀티 웰 플레이트 형식으로 포자 것은 허용한다.

<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> 이 프로토콜을 평가하기 위해, (전술 한 바와 같이) 멀티 웰 플레이트에서 세포를 포자로부터 얻어진 포자 효율은 형틀 (표 2)에 큰 볼륨을 사용 포자 세포를 비교 하였다. 멀티 웰 플레이트의 사용은 ~ 8​​0 %의 효율은 통상적으로 볼 수있는 플라스크에 포자 볼 때 높은 효율을 달성하지 않았다. (유리 비드 또는 교반 막대로 제공됨) 적합한 통기 멀티 웰 ?...

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.

효율적인 포자 형성<em&gt; 사카로 마이 세스 세레 비지에</em&gt; 96 멀티 웰 형식으로

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

10.0K 조회수.  University of Massachusetts Boston. Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

비디오: 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.

의 해부 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 ...

연구

생물학

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.

세계 S. cerevisiae의에서 organelle 형태를 연구하기 위해 높은 처리량 방법

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 분석은 신진 효모에서 스플라이세오솜 돌연변이체의 기질 특이적 민감도를 결정합니다.

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

유전학

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.

탠덤 질량 분광과 결합 된 액체 크로마토그래피를 사용하여 사카로미세스 Cerevisiae의 정량적 Metabolomics

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

생화학

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.

냉동 전자 단층 촬영을위한 사카로미세스 cerevisiae의 준비 및 Cryo-FIB 마이크로 매칭

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 분석을위한 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.

Barcoded 효모 게놈 라이브러리의 경쟁력 화면

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.

의 흐름 세포 계측법 기반 정화<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.

진화 방법에 감독<em&gt; 사카로 마이 세스 세레 비지에</em&gt; : 돌연변이 라이브러리 생성 및 검사

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 호흡과 발효 대사 분석을 배치 문화 지 수 성장 속도 론

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

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