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 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.
在芽殖酵母孢子形成进行了研究减数分裂1,基因重组2的机制,开发由细胞信号3的控制下,开发4的营养控制,转录期间提供见解生物学的许多方面,包括染色体分离的控制发展5调控,以及孢子形成6考试。孢子形成包括涉及的新的膜隔室的母细胞,随后用保护孢子壁6的沉积中形成的新的细胞分裂事件。这些研究,考察形成孢子的细胞经常采取迅速孢子酵母菌株SK1,其可以经受孢子形成的过程中约24小时以相对有效的方式7,8的优点。虽然对芽殖酵母产孢条件的优化已经被描述9-13,这些实验S于固体培养基或在孢子形成是利用培养管或瓶中大规模液体培养观察孢子形成。
在这里,我们描述了在96多井板格式形成孢子酵母的方法。我们发现,对于这种方法,曝气是用于同步和有效的孢子形成关键的,并且已经设计技术,以确保有足够的孢子形成的小体积多孔形式。在96多井板格式孢子允许使用高通量技术和多孔板格式,这种筛选使用平铺库14-16高拷贝抑制优化试剂进行筛选细胞。
在这里,我们提出了一个协议,用于在96多孔形式孢子SK1酵母。曝气是有效孢子形成,这需要使用任一搅拌棒或在每一个玻璃珠孔的关键。当细胞在96多孔板中形成孢子在没有任何一个珠或搅拌棒的振荡培养箱中,细胞不能有效地形成孢子。当细胞没有任何一个珠或在振荡培养箱搅拌棒相比,是在30℃下不搅拌( 表1形成孢子仅在孢子形成效率的少量增加被看见;在摇对10%17%放置在搅?…
The authors have nothing to disclose.
这项工作是由来自马萨诸塞州波士顿(LSH)和R15 GM86805从NIH大学(LSH)一约瑟夫·P·希利资助。 SMP是部分地由赛诺菲 – 健赞奖学金波士顿马萨诸塞州大学的支持。
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 |