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Method for separating saccharomyces cerevisiae haploids in ultrahigh-throughput manner on basis of flow cytometry

A flux separation and haploid technology, applied in the direction of microorganism-based methods, methods using spores, biochemical equipment and methods, etc., can solve the problems of low separation efficiency, time-consuming and labor-intensive, etc., and achieve the elimination of manual operations, The effect of avoiding diploid contamination

Inactive Publication Date: 2019-02-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method requires a lot of manual operations, and undispersed ascospores can also withstand heat treatment at 58°C, so the traditional method for isolating haploids of Saccharomyces cerevisiae has problems such as time-consuming, labor-intensive and low separation efficiency

Method used

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  • Method for separating saccharomyces cerevisiae haploids in ultrahigh-throughput manner on basis of flow cytometry
  • Method for separating saccharomyces cerevisiae haploids in ultrahigh-throughput manner on basis of flow cytometry
  • Method for separating saccharomyces cerevisiae haploids in ultrahigh-throughput manner on basis of flow cytometry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation of Saccharomyces cerevisiae spore suspension

[0030] (1) Diploid yeast strains were activated in YPD liquid medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L) at 30°C and 220rpm for 18h, then transferred to 50mL fresh In YPD liquid medium, culture at 30°C, 220rpm for 24h. Centrifuge at 4000rpm for 5min, collect the cells, wash twice with saline, spread on solid sporulation medium (potassium acetate 20g / L, agar powder 20g / L), and culture at 26°C for 3 days. Collect the sludge on the solid spore-forming medium and resuspend in 5 mL of saline to obtain untreated spore suspension.

[0031] (2) Add 2.5% (w / w) helicase (Sangon Bioengineering (Shanghai) Co., Ltd., Cat. No. A600870-0001) to the untreated spore suspension in step (1), and incubate at 30° C. for 30 minutes with shaking. A spore suspension treated with helicase was obtained.

[0032] (3) Add 35-45% (v / v) glass beads of 425-600 nm to the spore suspension treated in step (2), and sh...

Embodiment 2

[0033] The determination of the position of embodiment 2 spore cell group

[0034] Utilizing the characteristics that ascospores and vegetative cells are smaller than haploid spores, the area where haploid spore cells are located is determined according to the forward scattered light signal (FSC) of the flow cytometer ( figure 1 ). Respectively to the untreated sporulation sample (prepared and obtained in embodiment 1 step (1)), only the spore suspension sample (prepared and obtained in embodiment 2 step (1)) and helicase treatment gained with only helicase The spore suspension sample prepared by bead oscillation (prepared and obtained in embodiment 1 step (3)) carries out flow cytometric analysis, and the results are as follows: figure 1 shown. figure 1 In A, the untreated sample cell population is in the upper right area of ​​the figure; figure 1 In B, a part of haploid spores were isolated after the ascus wall was digested only by helicase treatment, and some cells were ...

Embodiment 3

[0035] Example 3 PI and FDA dyes stain the Saccharomyces cerevisiae spore suspension

[0036] Weigh 0.01g PI, and dilute to 10mL with PBS (pH 7.4) buffer solution. Dilute to 10 μg / mL to obtain PI fluorescent dye. Store at 4°C protected from light. Weigh 0.01g FDA and dilute to 5mL with acetone. Dilute to 2 μg / mL with PBS (pH 7.4) buffer solution to obtain FDA fluorescent dye. Store at 4°C protected from light. The spore suspension prepared by embodiment 1 was diluted to 10 5 ~10 6 For each cell / mL, mix 2 mL of spore suspension with equal volumes of 8 μg / mL PI dye and 4 μg / mL FDA dye respectively, and incubate at 4°C for 20 min in the dark.

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Abstract

The invention discloses a method for separating saccharomyces cerevisiae haploids in an ultrahigh-throughput manner on the basis of flow cytometry, and belongs to the technical field of high-throughput screening. The method has the advantages that ascospore treatment conditions are optimized, and accordingly the optimal saccharomyces cerevisiae spore suspension preparation conditions can be obtained; active spores and inactive cells in spore suspension are stained by PI (propidium iodide) and FDA (fluorescein diacetate) stain, stained samples are analyzed on the basis of flow cytometers, accordingly, the activity of saccharomyces cerevisiae spores can be distinguished, and the spores can be absolutely counted; the optimal spore suspension preparation conditions are established, and technologies for separating the saccharomyces cerevisiae haploids in the ultrahigh-throughput manner on the basis of the flow cytometers are created.

Description

technical field [0001] The invention relates to a method for separating Saccharomyces cerevisiae haploids based on ultra-high throughput of flow cytometry, and belongs to the technical field of high-throughput screening. Background technique [0002] Saccharomyces cerevisiae is widely used in fermentation industry, such as brewing industry, bioenergy industry and so on. At the same time, as a model organism, it has the advantages of clear genetic background, simple gene manipulation, and short cultivation period, and it is also widely used in basic research. In the field of fermentation industry, hybrid breeding is a traditional and efficient breeding method to improve the production performance of Saccharomyces cerevisiae. By crossing haploids with different excellent traits and then separating haploids, offspring haploids that integrate different excellent traits can be screened to improve industrial production performance. In the field of basic research, by crossing hap...

Claims

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Application Information

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IPC IPC(8): C12N3/00C12R1/865
CPCC12N3/00
Inventor 周景文陈坚曾伟主方芳堵国成夏小乐张伟平
Owner JIANGNAN UNIV
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