Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Summary

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

Abstract

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.

Introduction

Sporulation in the budding yeast has been studied to provide insights into many aspects of biology, including the control of chromosome segregation during meiosis¹, mechanisms of genetic recombination², the control of development by cell signaling³, nutritional control of development⁴, the transcriptional regulation of development⁵, and the examination of spore formation⁶. Spore formation includes a novel cell division event involving the formation of new membrane compartments within the mother cell followed by the deposition of a protective spore wall⁶. These studies that examine sporulating....

Protocol

1. Preparing for Sporulation

Note: The media described in this protocol are made using standard recipes and methods^13,17. Table 1 gives the formulation for 1 L of the various media used in this protocol.

Bacto Peptone

Yeast Extract

Bacto Agar

Dextr....

Results

To assess this protocol, sporulation efficiencies obtained from sporulating cells in multiwell plates (as described above) were compared to cells sporulated using larger volumes in flasks (Table 2). The use of multiwell plates did not achieve the high efficiency seen when sporulating in flasks, where ~80% efficiency can be routinely seen. Sporulating in multiwell plates with proper aeration (provided by glass beads or stir bars) can achieve sufficient sporulation efficien.......

Discussion

Here we present a protocol for sporulating SK1 yeast in a 96 multiwell format. Aeration is key for efficient sporulation, which requires the use of either a stir bar or a glass bead in each well. When cells are sporulated in a 96 multiwell plate in a shaking incubator without either a bead or a stir bar, cells do not sporulate efficiently. Only a small increase in sporulation efficiency is seen when cells are sporulated without either a bead or a stir bar in a shaking incubator, compared to being at 30 °C without ag.......

Materials

List of materials used in this article

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