עבודה זו נתמכה על ידי מענק ג&#39;וזף פ הילי מאוניברסיטת מסצ&#39;וסטס בבוסטון (LSH) ו R15 GM86805 מ- NIH (LSH). SMP הוא נתמך בחלקו על ידי עמיתי Sanofi-ג&#39;נזיים באוניברסיטת מסצ&#39;וסטס בבוסטון.

1. הכנות לקראת נביגה הערה:. התקשורת המתוארת בפרוטוקול זה מבוצע באמצעות 13,17 מתכונים ושיטות תקן הטבלה 1 נותנת את הניסוח עבור 1 ליטר של התקשורת השונה בשימוש בפרוטוקול זה. <table border="1" cellpadding="0" cellspacing="0" height="180" width="469" style=";text-align:right;direction:rtl"><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;"> bacto Peptone </td><td style="width: 50px;"> תמצית שמרים </td><td style="width: 50px;"> bacto אגר </td><td style="width: 60px;"> דקסטרוז </td><td style="width: 70px;"> אשלגן יצטט </td><td style="width: 60px;"> גליצרול </td><td style="width: 60px;"> DDH 2 O </td></tr><tr height="15"><td height="15" style="height:20px;"> צלחות YPG </td><td> 20 גרם </td><td> 10 גרם </td><td> 20 גרם </td><td> - </td><td> - </td><td> 30 מ&quot;ל </td><td> 970 מ&quot;ל </td></tr><tr height="15"><td height="15" style="height:20px;"> צלחות YPD </td><td> 20 גרם </td><td> 10 גרם </td><td> 20 גרם </td><td> 20 גרם </td><td> - </td><td> - </td><td> 1,000 מ&quot;ל </td></tr><tr height="15"><td height="15" style="height:20px;"> נוזלי YPD </td><td> 20 גרם </td><td> 10 גרם </td><td> - </td><td> 20 גרם </td><td> - </td><td> - </td><td> 1,000 מ&quot;ל </td></tr><tr height="15"><td height="15" style="height:20px;"> נוזל YPA </td><td> 20 גרם </td><td> 10 גרם </td><td> - </td><td> - </td><td> 20 גרם </td><td> - </td><td> 1,000 מ&quot;ל </td></tr><tr height="15"><td style="height: 20px; width: 80px;"> תקשורת נביגה </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 10 גרם </td><td> - </td><td> 1,000 מ&quot;ל </td></tr></tbody></table> טבלה 1:. ניסוחים מדיה סכומים ניתנים 1 ליטר של התקשורת. פרטים על תקשורתניתן למצוא מרכיבים של חומרי הטבלה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אם כל תאי Sporulating יהיו גנוטיפ הזהה, בצע הליך זה: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> זן שמרים ההפשרה לשמש לצלחת YPG (YPGlycerol). לגדול שמרים במשך 12-16 שעות על 30 מעלות צלזיוס. חשוב לציין, תאי sK1 עומדים גם sporulate, כך זני sK1 אסור להשאיר יותר מדי זמן על צלחות YPG, או שהם עשויים sporulate על הצלחת. הערה: זן טרי מופשר גדל על YPG הוא העדיף, כמו ניצול של גליצרול כמקור פחמן דורש זרחון חמצוני, ובכך בוחר עבור תאים בעלי הדנ&quot;א המיטוכונדרי פונקציונלי. יש sK1 תאים נטייה לאבד הדנ&quot;א המיטוכונדרי פונקציונלי ולהיות petites. הדנ&quot;א המיטוכונדרי פונקציונלי דרוש נביגה. </li><li style=";text-align:right;direction:rtl"> שמרים Streak מהצלחת YPG לצלחת YPD (YPDextrose) עבור מושבות אחת. לגדול שמרים על 24-28 שעות צלחת YPD על 30 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> אחרי בערך 36-48 שעות של צמיחה על תקשורת המוצקה YPD, להתחיל 25 מיליליטר תרבות YPD באמצעות מושבה אחתמהצלחת YPD. לגדול בתרבות זו עד תאים על OD 600 = 4.0 ל -7.0. בצע פעולה זו על 30 מעלות צלזיוס לילה חממה רועד, רועד ב 220 סל&quot;ד. </li><li style=";text-align:right;direction:rtl"> מתרבות YPD, השתמש כמות מתאימה של תאים להתחיל 1,000 מ&quot;ל של YPA (YPAcetate) תרבות הוא OD 600 = 0.1 עבור כל צורך בצלחת 96 באר בסעיף 2. לגדול השמרים 30 ° C רועד חממה (רועד ב 220 סל&quot;ד) עד התרבות OD 600 היא בין 1.3 1.5. הערה: לחסן 100 מ&quot;ל של תרבות YPA עבור 96 כל צלחת multiwell שיוקרנו בסעיף 2. לדוגמה, אם ההקרנה ארבע 96 צלחות multiwell, 400 מ&quot;ל של תרבות YPA יהיה צורך. החל נפח תוספת 10% בתרבות YPA יש צורך להגן מפני איבוד מסת אוויר. בדרך כלל, זה ייקח בין 12-16 שעות עבור תאים להגיע OD המתאים 600; את משך הזמן עשוי להשתנות תלוי גנוטיפ של זן השמרים. YPA הוא התקשורת presporulation. Pregrowthב תצטט עוזר להשרות ביטוי של גנים (כגון IME1) דרושי נביגה יעיל סינכרוני 6. </li><li style=";text-align:right;direction:rtl"> ספין 1,800 XG בצנטריפוגה בראש הטבלה במשך 5 דקות עד גלולה התאים הגדלים YPA. לשטוף את התאים 1X עם 0.5 נפח של מים ללא יונים autoclaved סטרילי. ספין שוב, וזורקים לשטוף. Resuspend תאי תקשורת נביגה נפח 1x. המשך לסעיף 2. </li></ol></li><li style=";text-align:right;direction:rtl"> אם באמצעות תאים עבור נביגה כי הם של גנוטיפים שונים, (כלומר, הפך עם פלסמידים שונים או המכילים מוטציות שונות, בדרך כלל מתקבל ומאוחסנים כמו מניות קפוא), תאי מערך של גנוטיפים שונים לפורמט multiwell 96. לאחר מכן, בצע את התהליך הבא: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש Frogger 96 שיניים סטרילי להעביר תאים ממניות קפוא מופשר ב 96 צלחת multiwell (ים) על גבי מדיה YPG מוצק. לגדול לילה שמרים על 30 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> באמצעות Frogger 96 שיניים סטרילי, להעביר תאי תקשורת מוצקה YPG גבי מדיה מוצקה YPD או סלקטיביתתקשורת מוצקה (כלומר, תקשורת מינימאלית סינטתית חסרת חומצות אמינו, אם התאים מכילים פלסמידים הדורשים מבחר). לגדול שמרים ב 30 מעלות צלזיוס עד מושבות נוצרות עבור כל זן, בדרך כלל 1 יום על YPD ו -2 ימים אם התקשורת סלקטיבית היא הכרחית. </li><li style=";text-align:right;direction:rtl"> Aliquot 1.2 מיליליטר של תקשורת הנוזלת YPD או תקשורת הנוזלת סלקטיבית לבאר כל צלחת multiwell 96 עמוק 2 מיליליטר. העברת תאים מן התקשורת מוצקה לתוך תקשורת נוזלית באמצעות פרוגר, מכסים עם מכסה פלסטיק מוחזק במקום על ידי קלטת, ולגדול לילה 30 ° C שייקר, רועד ב 220 סל&quot;ד. הערה: כדי למזער זיהום, אטמים באמצעות מכסי הפלסטיק מתוך צלחת הפטר מלבני. החזק מכסה במקום באמצעות נייר דבק, כך זרימת האוויר מוגבלת מינימאלית. </li><li style=";text-align:right;direction:rtl"> צלחות צנטריפוגה ב 1,800 XG במשך 5 דקות, באמצעות צנטריפוגות דלי מתנדנד עם מתאמים כדי להכיל 96 צלחות היטב. יוצקים את התקשורת מתאי; להוסיף 500 μl YPA זה היטב resuspend התאים pelleted ב YPA. כיסויעם מכסה פלסטיק מוחזק במקום על ידי קלטת. </li><li style=";text-align:right;direction:rtl"> לגדל תאים עבור 15 שעות ב 30 מעלות צלזיוס חממה רועדת, רועד ב 220 סל&quot;ד. המשך לסעיף 2. </li></ol></li></ol> 2. תאים Sporulating במתכונת 96 multiwell <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להוסיף אחד מהם 3 מ&quot;מ חרוז זכוכית מוצקה סטרילי או בר ומערבבים מגנטי סטרילי 5 מ&quot;מ x 2 מ&quot;מ לבאר כל צלחת 96 גם עם 1.3 מ&quot;ל בארות. (ראה תוצאות נציג להלן דיון בשאלה האם להשתמש חרוז או בר ומערבבים.) </li><li style=";text-align:right;direction:rtl"> אם כל התאים שיוקרנו הם מאותו גנוטיפ, ללכת 2.2.1. אם תאים שיוקרנו הם של גנוטיפים שונים, ללכת 2.2.2. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קח התאים משלב 1.1.5 ו aliquot 500 μl של תאים בתוספת התקשורת לבאר כל 96 הצלחת multiwell. מכסים עם מכסה פלסטיק מוחזק במקום על ידי קלטת. המשך 2.3. </li><li style=";text-align:right;direction:rtl"> תאי ספין הנמצאים YPA מ 1.2.4 1,800 XG במשך 5 דקות, באמצעות צנטריפוגות דלי מתנדנד עם מתאמים כדי להכיל 96 צלחות היטב. יוצקים שלמדיה מתאי f. הוסף 500 μl של התקשורת נביגה זה היטב resuspend התאים pelleted בתקשורת נביגה. מכסים עם מכסה פלסטיק מוחזק במקום על ידי קלטת. המשך 2.3. </li></ol></li><li style=";text-align:right;direction:rtl"> אם באמצעות חרוז זכוכית, דגימות מקום חממה רועדת ב 30 ° C ולנער ב 220 סל&quot;ד במשך נביגה. אם אתם משתמשים במכשיר בר ומערבבים מגנטיים, דגימות מקום בצלחת ומערבבים בתוך חממת 30 מעלות צלזיוס במשך נביגה. הערה: המהירות והמערבבים המדויקת עבור הברים ומערבבים המגנטיים אינה חשובה מאוד, כל עוד כל הברים נעים במרץ ואת התרבות מוגזים. </li><li style=";text-align:right;direction:rtl"> כדי לעקוב אחר התקדמות דרך נביגה, לסגת דגימות מתרבות sporulating להדמיה, להוסיף 6 μl של תאים מן התרבויות sporulating 24 μl של התקשורת נביגה בצלחת coverslip 96 באר (1: 5 דילול). השאירו תאים להסתפק 5 דקות לפני בחינת התאים באמצעות מיקרוסקופ הפוכה. הערה: תאי sK1 סוג ברים, נבגים בוגרים קומפקטיים יהוו ידי 36 שעות לאחר העברת מדיה נביגה. התקדמות דרך נביגה ניתן לנטר באמצעות מיקרוסקופ באמצעות סמנים ניאון, כגון סמן גרעיני (כלומר, Htb2-mCherry 18) לבחון את חלוקת meiotic או סמן קרום prospore (כלומר, G20 19) לבחון morphogenesis נבג. לחלופין, היווצרות נבגי refractile ניתן לראות באמצעות מיקרוסקופ Nomarski דסק&quot;ש החל כ -12 שעות לאחר העברה למדיה נביגה. </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+and+negative+regulation+of+a+SNARE+protein+by+control+of+intracellular+localization.">Positive and negative regulation of a SNARE protein by control of intracellular localization.</a> Mol. Biol. Cell. 15 (4), 1802-1815 (2004).</li><li>Huang, L. S., Doherty, H. K., Herskowitz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Smk1p+MAP+kinase+negatively+regulates+Gsc2p,+a+1,3-beta-glucan+synthase,+during+spore+wall+morphogenesis+in+Saccharomyces+cerevisiae.">The Smk1p MAP kinase negatively regulates Gsc2p, a 1,3-beta-glucan synthase, during spore wall morphogenesis in Saccharomyces cerevisiae.</a> Proc. Natl. Acad. Sci. USA. 102 (35), 13431-13436 (2005).</li></ol>

The authors have no competing financial interests.

כאן אנו מציגים פרוטוקול sporulating שמרים sK1 במתכונת multiwell 96. אוורור הוא מפתח עבור נביגה יעילה, אשר מחייבת שימוש או בר ומערבב או חרוז זכוכית בכל טוב. כאשר תאים התנבג בצלחת multiwell 96 בתוך חממה רועדת בלי שאחד חרוז או ומערבבים ברים, תאים אינם sporulate ביעילות. רק עלייה קטנה יעילות נבי?...

נביגה ב השמרים ניצני נחקרה לספק תובנות היבטים רבים של הביולוגיה, כולל שליטה על הפרדה כרומוזום במהלך המיוזה 1, מנגנוני שחלוף 2, שליטה של פיתוח על ידי התא איתות 3, שליטה התזונתי של פיתוח 4, תעתיק הסדרת פיתוח 5, ובחינת יצירת הנבגים 6. יצירת נבגים כוללת אירוע חלוקת תא רומן מעורב ההיווצרות של תאי קרום חדשים בתוך תא אמא ואחריו בתצהיר של קיר נבג מגן 6. מחקרים אלה הבוחנים תאים sporulating מנצלים לעתים קרובות זן שמרים sporulating במהירות sK1, אשר יכול לעבור תהליך של נביגה בערך 24 שעות בצורה יעילה יחסית 7,8. למרות אופטימיזציה של תנאי נביגה בשביל נבגי שמרים תוארו 9-13, אלה לביצוע הניסויs בחן נביגה על התקשורת מוצק או נוזלי בתרבויות בקנה מידה גדול יותר שבו נביגה מתבצעת באמצעות צינורות תרבות או צלוחיות. כאן אנו מתארים שיטה sporulating שמרים במתכונת צלחת multiwell 96. אנו מוצאים כי בשיטה זו, אוורור הוא קריטי עבור נביגה סינכרוני ויעיל, פיתחו טכניקות כדי להבטיח נביגה מספיק בפורמט multiwell קטן-נפח. Sporulating במתכונת צלחת multiwell 96 מאפשר לתאים להיות מוקרנים באמצעות טכניקות תפוקה גבוהה ריאגנטים מותאם במיוחד בפורמט צלחת multiwell, הקרנה כזה מדכאי גבוהה עותק באמצעות ספריית רעפים 14-16.

<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> כדי להעריך בפרוטוקול זה, יעילות נביגה המתקבלת sporulating תאי צלחות multiwell (כמתואר לעיל) הושוותה תאים התנבג באמצעות כמויות גדולות יותר צלוחיות (טבלה 2). השימוש צלחות multiwell לא להשיג את היעילות הגבוהה לראות כאשר sporulating צלוחיות, שבו ~ 80% יעילות ניתן ?...

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.

נביגה יעילה של<em&gt; שמר האפייה</em&gt; בתוך פורמט 96 multiwell

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

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Research

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Biology

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

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

Video: 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.

Dissection של 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 ...

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.

שיטת תפוקה גבוהה ברחבי העולם כדי ללמוד את המורפולוגיה של אברון cerevisiae ס

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.

מבחני ACT1-CUP1 קובעים את הרגישויות הספציפיות למצע של מוטנטים שחבורים בשמרים ניצנים

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

Genetics

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.

מטבולומיה כמותית של Saccharomyces Cerevisiae באמצעות כרומטוגרפיה נוזלית בשילוב עם ספקטרומטריית מסה דו-מושבית

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

Biochemistry

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.

הכנה ומיקרו-מכונות קריו-FIB של סרביסיה Saccharomyces עבור טומוגרפיה קריו-אלקטרון

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

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.

מסכי הגנום תחרותי של ספריות שמרים המבורקדים

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

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

כושר ניתוח כמותי של זרימת עבודה

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

תהליך המפלצת הירוקה לדור של זני שמרי נשיאת מחיקות Gene מרובות

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

הקרנה תפוקה גבוהה של חלבון מבוססת ירושה cerevisiae ס

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

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.

שמר האפייה קינטיקה גידול אקספוננציאלי בתרבות אצווה כדי לנתח את חילוף החומרים הנשימה ואת Fermentative

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

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