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Method for screeningabamectin high-yield strain in high throughput based on flow cytometry

A technology of flow cytometry and abamectin, applied in the field of high-throughput screening of high-yielding abamectin strains, can solve the problems of long screening cycle, dead cells, heavy workload, etc., and achieve sorting speed and screening efficiency The effect of improving, increasing growth rate, and solving growth difficulties

Inactive Publication Date: 2016-12-14
JIANGNAN UNIV
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AI Technical Summary

Problems solved by technology

[0003] The current classical mutagenesis combined with high-throughput screening method may lead to the failure of subsequent culture due to the existence of a large number of dead cells in the classical mutagenesis. At the same time, the strains after the classical mutagenesis must be further isolated, purified, and expanded for subsequent enzyme labeling. There are problems such as long screening period and heavy workload in the early stage for high-throughput screening such as plate screening

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1ARTP mutagenesis or other mutagenesis methods obtain different active spores

[0042] The slant strain of the starting strain Streptomyces avermitilis was cultured at 28°C for 5 days until the spores matured and the slant colonies were off-white. Scrape an appropriate amount of mature slant spores into a test tube filled with 2mL PBS buffer solution, vibrate with a shaker to make a bacterial suspension, and filter it with sterile filter paper. Draw 10 μL of a suitable concentration of bacterial suspension and evenly spread it on the surface of a sterile slide, place the slide on the stage, and use the ARTP mutagenesis instrument for mutagenesis. Conditions: The incident power is 100W, the helium flow rate is 10SLM, and spores with different activities are obtained by irradiating for a certain period of time. Other mutagenesis methods to obtain spores with different activities are also applicable.

Embodiment 2PI

[0043] Embodiment 2PI staining agent is to Streptomyces avermitilis spore staining

[0044]After the sample is processed, use sterile tweezers to place the slide into an EP tube filled with PBS (pH 7.4) buffer solution, and shake thoroughly for 1 min to completely elute the bacteria attached to the slide into the liquid PBS. Filter with filter paper, centrifuge at 5000×g for 5 min, discard the supernatant, and add PBS to resuspend. Repeat this operation step 2 to 3 times to obtain pure Streptomyces avermitilis spore suspension. Dilute the pure S. avermitilis spore suspension to a concentration of 10 with PBS 5 ~10 6 spores / mL. Take an equal volume of Streptomyces avermitilis spore suspension and mix with 10 μg / mL PI stain, and place in a 4°C refrigerator in the dark for 15 minutes to incubate.

[0045] Example 3 Flow Cytometry Detection and Sorting

[0046] Detection should establish a control test to remove non-specific binding or background. A sample not labeled with a...

Embodiment 4

[0047] Embodiment 4 microplate reader detects

[0048] According to the fact that abamectin has the maximum light absorption at 240nm, but the blank culture medium has no absorption value at 240nm, the detection of abamectin by microplate reader was selected as the primary screening method. Configure a standard with a certain concentration gradient and detect it with a microplate reader to obtain a standard curve equation y=0.01782x+0.06191, R 2 =0.99715, x is the standard substance concentration (μg / mL), y is OD 240nm , the standard concentration range is 0-210μg / mL. The pre-fermentation experiment in the 48-well plate showed that the titer of avermectin was around 1000 μg / mL. Draw 50 μL of the culture solution in the 48 deep-well plate, dilute the abamectin with ethanol to the linear range of the standard curve, centrifuge at 4000r / min for 15min after ultrasonic extraction, take 200μL of the supernatant on a 96 microplate plate, and use a microplate reader Measure the abs...

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Abstract

The invention discloses a method for screening abamectin high-yield strain in high throughput based on a flow cytometry, belonging to the technical field of high-troughput screening. The method comprises the following steps: performing PI dyeing method on mutated spores with different activities, correctly distinguishing a spore area with activity by regulating and optimizing various parameters such as FSC, SSC and voltage threshold, and excluding the interference mixed with the background in flow cytometry detection due to oversmall volume of spores. The screened live spores are subjected to a solid-liquid combined high-throughput culturing scheme to effectively improve the growth rate of an abamectinpore plate. According to the invention, the abamectin with activity can be screened based on the flow cytometry, and a novel and effective manner is provided for the high-throughput screening of abamectin.

Description

technical field [0001] The invention relates to a method for high-throughput screening of high-throughput abamectin-producing strains based on flow cytometry, and belongs to the technical field of high-throughput screening. Background technique [0002] Abamectin, as a secondary metabolite of Streptomyces avermitilis, has a complex and huge network in its metabolism. Although the whole genome sequencing of abamectin has been completed, its biosynthesis process is complex, regulated at different levels, and its metabolic pathway has not been clearly elucidated. Molecular breeding methods are expensive and complicated. In order to speed up the breeding speed, it is necessary to establish a simple, fast and accurate detection method. High-throughput screening technology is based on traditional screening technology, organically combining biochemistry, modern biology, computer, automatic control and other high-tech into a new high-automation mode. Compared with traditional rand...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/01C12N1/20C12N1/02C12R1/465
CPCC12N15/01C12N1/02C12N1/20
Inventor 周景文陈坚陈玥王得明曹晓梅张庆辉王丽丽堵国成
Owner JIANGNAN UNIV
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