Work in the author&rsquo;s laboratory is funded by the Canadian Institutes of Health Research, the Canada Foundation for Innovation, the Natural Sciences and Engineering Research Council and Concordia University.

Construction of diploid strains 
Protocol for production of diploids by mating
<ol>
	<li>The initial strains should be struck out on YPD, or selective medium if necessary, to produce single colonies. The two haploid strains should be of opposite mating type (ie. MATa and MAT&#945;).</li>
	<li>Using a sterile inoculation loop pick a small portion of a colony from one of the two haploid strains. On a new YPD plate streak the loop once in a straight line across the plate. Mark the direction of the streak with an arrow on the bottom of the plate.</li>
	<li>Repeat this for the second strain on the same YPD plate. This time streak the colony perpendicular to the first, crossing the first streak with the second. Mark the direction of the second streak and note the area where the second streak crosses the first. This area will be where diploids will be produced.</li>
	<li>Incubate the YPD plate at 30&#176;C overnight.</li>
	<li>Replica plate the YPD plate onto selective media that will selectively permit growth of the newly-formed diploids.</li>
	<li>Pick a single colony if possible, or a small portion of the diploid streak and streak onto the same selective medium to generate single colonies.</li>
</ol>
Sporulation and digestion of asci 
Protocol
<ol>
	<li>Pick single colonies from the plate above and make small (1cm x 1cm) patches on sporulation (SPO) plates and incubate at 30&#176;C for 3-7 days to sporulate.</li>
	<li>Sporulation can be checked by looking at cells from each patch under a light microscope. Place cells on a microscope slide in 5-10 &#956;l of water, place a coverslip on top of the droplet, and look for the presence of asci. If asci are not easily found then put back in the incubator.</li>
	<li>If there is effective sporulation in one of the patches, prepare a sterile 15mL conical tube with 100&#956;L of tetrad juice.</li>
	<li>Pick cells up from this sporulation patch with a sterile 3mm inoculation loop (cells should cover &#188; of the loop) and place in the tetrad juice. Gently swirl the loop to allow the cells to fall off the loop and mix with the solution.</li>
	<li>Leave in tetrad juice (containing zymolyase) for 4-10mins (depending on the concentration and activity of zymolyase and the strain itself).This allows for digestion of the cell wall surrounding the ascospores.</li>
	<li>Mark the top and bottom of the inoculum region on a YPD plate.</li>
	<li>Optional. Reaction can be stopped by adding 1mL of ice cold sterile dH2O. Allow the cells to settle for 5mins. Remove 1mL from the supernatant then mix lightly by hand and keep on ice.</li>
	<li>Take 5&#956;L of digested cells from step 5 and gently place drops in a line in the inoculum region of the pre-marked YPD plate.</li>
	<li>Use a sterile 3mm inoculation loop to very gently spread the drops 1-3 times in a line while barely making contact with the agar.</li>
	<li>Allow the liquid to absorb into the agar (If step 7 is omitted, this is when the digestion of the outer cell wall will be reduced or stop). Proceed to the dissection microscope.</li>
</ol>
Dissection of Asci 
Protocol
Note that the following protocol is described for a Singer MSM System 200 micromanipulation microscope. This protocol can be followed with slight modifications if using a manual micromanipulator such as a Zeiss micromanipulator MR.
<ol>
	<li>Focus on a region of the plate where half of the field contains cells and half is empty. Slowly bring the needle into focus such that it touches the empty region of the field. This will ensure that the needle does not contain cells from a previous use. Contact between the needle and the agar is seen as a dark black circle that slowly comes into focus.</li>
	<li>Go to the matrix at position A1 and make a mark by piercing the agar again. This is to help determine the orientation of the plate if it is taken off the microscope before completion.</li>
	<li>Switch to the search mode. Locate an asci. Preferably choose asci which are in areas where the cells are well spread out. Avoid areas with cell clusters. The four ascospores from an asci should still be attached to each other. Two ascospores may be slightly larger than the other two. Avoid picking asci where one ascospore is loose and not connected to the rest of the ascospores.</li>
	<li>Pick up an ascus with the needle. Slowly bring the needle up to the plate. When the shadow of the microneedle is seen, line this up with the ascus to be picked. Again approach the needle until the black outline of a circle (the tip of the needle) is seen. Touch the tetrad with this circle and then quickly pull the needle away. Make sure all four ascospores from an ascus have adhered to the needle and that no additional cells were gathered on the microneedle that are not part of this ascus.</li>
	<li>Pull the microneedle far away from the plate and go to the matrix (for the first ascus, go to position A1). Take note of each position where an ascus is relocated. Place the ascus down by touching the plate with the microneedle and pulling away until all four ascospores come off the microneedle. Gentle tapping of the needle arm or the platform holding the YPD plate is often useful at this step.</li>
	<li>Repeat steps 3-5 and place asci down at consecutive positions on the matrix until the desired number of asci have been picked.</li>
	<li>Go back to each ascus and break the four cells apart by teasing with the needle, tapping the platform gently or a combination of both. Pick up the cells leaving one behind at a new position in the matrix until each ascospore in each ascus is separated in rows of four.</li>
	<li>Place the YPD plate in an incubator at 30&#176;C for several days to observe the growth pattern.</li>
	<li>Replica plate the dissected cells to selective medium if necessary.</li>
</ol>
Relocation of Zygotes 
Protocol
<ol>
	<li>Streak out two haploid strains with opposite mating types on the appropriate medium to obtain single colonies.</li>
	<li>Mate these two strains as described above and allow to grow from 4 to 6 hours (or overnight if necessary).</li>
	<li>Check for zygotes under a light microscope. Zygotes will appear as dumbbell-shaped cells either with or without a bud.</li>
	<li>Pick cells up with a 3mm inoculation loop (cells should cover &#188; of the loop) and place gently in 100&#956;L of sterile dH2O.</li>
	<li>Mark the top and bottom of the inoculum region on a YPD plate.</li>
	<li>Take 5&#956;L and gently place drops in a line in the inoculum region of the pre-marked YPD plate.</li>
	<li>Use a 3mm inoculation loop to spread the drops in a line very gently while barely making contact with the agar.</li>
	<li>Allow the liquid to absorb into the agar. Proceed to the dissection microscope.</li>
	<li>Focus on a region of the plate where half of the field contains cells and half is empty. Slowly bring the needle into focus such that it touches the empty region of the field. This will ensure that the needle does not contain cells from a previous use. Contact between the needle and the agar is seen as a dark black circle that slowly comes into focus.</li>
	<li>Go to the matrix at position A1 and make a mark by piercing the agar again. This is to help determine the orientation of the plate if it is taken off the microscope before completion.</li>
	<li>Switch to the search mode. Locate a zygote.</li>
	<li>Pick up a zygote with the needle.</li>
	<li>Place zygotes individually on the matrix.</li>
	<li>Allow zygotes to grow for several days.</li>
	<li>Replica plate the zygotes onto the appropriate selective medium.</li>
</ol>
Representative Results
<img alt="" src="/files/ftp_upload/1146/1146fig1.jpg" /> 
Figure 1: The mating of two haploid strains of opposite mating types generates a diploid strain.
<img alt="" src="/files/ftp_upload/1146/1146fig2.jpg" /> 
Figure 2: (A) A YPD plate with thre results of a mating between a temperature sensitive strain (with a leu2 auxotrophy) and a wild type strain (LEU2). Digested cells are spread in the inoculum region on the left where thick growth between the two black lines is seen. Asci that have been dissected grow evenly spaced in the matrix on the right. (B) The plate in (A) was replicated onto an SD -Leucine drop-out plate. Note the 2:2 growth for the LEU2 marker indicating a validated ascospore was dissected. (C) The plate in (A) was replicated onto a YPD plate and incubated at 37&#176;C. Note the 2:2 growth that further validates that an ascospore was dissected.

<ol>
	<li>Sherman, F., Hicks, J., Guthrie, C., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulation+and+Dissection+of+Asci.">Micromanipulation and Dissection of Asci.</a> Guide to Yeast Genetics and Molecular Biology. , 21-37 (1991).</li></ol>

The authors have nothing to disclose.

Yeast dissection is a useful tool to select new strains with desired markers. When determining the theoretical growth pattern of four ascospores it is important to look at the genotype of the vegetative cell. If a plasmid is present, one must know if it is single, low or high copy as this will affect which ascospore(s) receive the plasmid and will affect predictions and conclusions regarding the strains being manipulated.
Certain strains can sporulate better than others producing many ascospor...

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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Singer MSM System 200</td>
<td>Microscope</td>
<td>Singer Instruments</td>
<td>NA</td>
<td>&nbsp;</td>
<tr>
<td>D-sorbitol</td>
<td>Reagent</td>
<td>BioShop</td>
<td>SOR508</td>
<td>&nbsp;</td>
<tr>
<td>Tris</td>
<td>Reagent</td>
<td>BioShop</td>
<td>TRS001</td>
<td>&nbsp;</td>
<tr>
<td>Zymolyase 100T</td>
<td>Reagent</td>
<td>Seikagaku Corporation</td>
<td>120493</td>
<td>&nbsp;</td>
<tr>
<td>Yeast extract</td>
<td>Reagent</td>
<td>BioShop</td>
<td>YEX401</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>BioShop</td>
<td>PEP 403</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>BioShop</td>
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<td>&nbsp;</td>
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<td>Reagent</td>
<td>BioShop</td>
<td>YNB406</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>BioShop</td>
<td>TRP402</td>
<td>&nbsp;</td>
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<td>Sodium chloride</td>
<td>Reagent</td>
<td>BioShop</td>
<td>SOD002</td>
<td>&nbsp;</td>
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<td>Potassium acetate</td>
<td>Reagent</td>
<td>BioShop</td>
<td>POA 301</td>
<td>&nbsp;</td>
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<td>Agar</td>
<td>Reagent</td>
<td>BioShop</td>
<td>AGR003</td>
<td>&nbsp;</td>
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<td>Adenine sulfate</td>
<td>Reagent</td>
<td>BioShop</td>
<td>ADS201</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>BioShop</td>
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<td>&nbsp;</td>
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<td>Reagent</td>
<td>BioShop</td>
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<td>Reagent</td>
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<td>&nbsp;</td>
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<td>Reagent</td>
<td>BioShop</td>
<td>MET 222</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>BioShop</td>
<td>TYR 333</td>
<td>&nbsp;</td>
<tr>
<td>L-isoleucine</td>
<td>Reagent</td>
<td>BioShop</td>
<td>ISO 910</td>
<td>&nbsp;</td>
<tr>
<td>L-valine</td>
<td>Reagent</td>
<td>BioShop</td>
<td>VAL 201</td>
<td>&nbsp;</td>
<tr>
<td>L-lysine</td>
<td>Reagent</td>
<td>BioShop</td>
<td>LYS 101</td>
<td>&nbsp;</td>
<tr>
<td>L-phenylalanine</td>
<td>Reagent</td>
<td>BioShop</td>
<td>PHA 302</td>
<td>&nbsp;</td>
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<td>L-glutamic acid</td>
<td>Reagent</td>
<td>BioShop</td>
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<td>Reagent</td>
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<td>&nbsp;</td>
<tr>
<td>L-leucine</td>
<td>Reagent</td>
<td>BioShop</td>
<td>LEU 222</td>
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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.

Watch this Scientific Journal Video about Dissection of Saccharomyces Cerevisiae Asci at JoVE.com

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 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 ...

dissection-of-saccharomyces-cerevisiae-asci

Research

JoVE Journal

Biology

18.5K Aufrufe.  Concordia University. Mikromanipulation von Hefezellen ist für meiotischen genetische Analyse oder diploiden Zygoten wählen erforderlich. Diese Mikromanipulationen werden mit dem Mikronadel einer Dissektionsmikroskop durchgeführt. Die Mikronadel wird verwendet, um Zellen zu verlagern und wird durch einen Mikromanipulator, die mit verschiedenen Graden der Automatisierung gesteuert werden.

Serie: Dissection of Saccharomyces Cerevisiae Asci

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

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.

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

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

Genetics

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.

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

Biochemistry

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

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.

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

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S.&#160;cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

The CRISPR/Cas12a system in combination with a single crRNA array enables efficient multiplex editing of the S.&#160;cerevisiae genome at multiple loci simultaneously. This is demonstrated by constructing carotenoid producing yeast strains which are subsequently used to create yeast pixel art.

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ...

Bioengineering

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Microbes typically live in communities. The spatial organization of cells within a community is believed to impact the survival and function of the community1. Optical sectioning techniques, including confocal and two-photon microscopy, have proven useful for observing spatial organization of bacterial and archaeal communities2,3. A combination of confocal imaging and physical sectioning of yeast colonies has revealed internal organization of cells4. However, direct optical sectioning using confocal or two-photon microscopy has been only able to reach a few cell layers deep into yeast colonies. This limitation is likely because of strong scattering of light from yeast cells4.
Here, we present a method based on fixing and cryosectioning to obtain spatial distribution of fluorescent cells within Saccharomyces cerevisiae communities. We use methanol as the fixative agent to preserve the spatial distribution of cells. Fixed communities are infiltrated with OCT compound, frozen, and cryosectioned in a cryostat. Fluorescence imaging of the sections reveals the internal organization of fluorescent cells within the community.
Examples of yeast communities consisting of strains expressing red and green fluorescent proteins demonstrate the potentials of the cryosectioning method to reveal the spatial distribution of fluorescent cells as well as that of gene expression within yeast colonies2,3. Even though our focus has been on Saccharomyces cerevisiae communities, the same method can potentially be applied to examine other microbial communities.

We present a protocol for freezing and cryosectioning yeast communities to observe internal patterns of fluorescent cells. The method relies on methanol-fixing and OCT-embedding to preserve the spatial distribution of cells without inactivating fluorescent proteins within a community.

Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

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.

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

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

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

Dissection of the Endolymphatic Sac from Mice

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the ...