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A method for high-throughput screening of aureomycin high-producing strains based on flow cytometry

A flow cytometry, high-throughput technology, applied in the field of high-throughput screening of aureomycin high-yield strains, can solve the problems of a large number of dead cells, uncultivable cultivability, long screening cycle, etc., to achieve increased growth rate, sorting Improvement of speed and screening efficiency, effect of solving growth difficulties

Active Publication Date: 2019-05-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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. High-throughput screening such as plates, but there are problems such as long screening cycle

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 ARTP mutagenesis or other mutagenesis to obtain different active spores

[0041] The starting strain Streptomyces aureus slant strain was cultured at 34°C for 4 days until the spores matured, and the slant colony was mouse gray. Scrape an appropriate amount of mature slant spores into a test tube filled with 2mL of PBS buffer solution, vibrate with a shaker to make a bacterial suspension, and filter 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. The spores with different activities were obtained by irradiating for a certain time respectively. Other physical and chemical mutagenesis can also obtain spores with diffe...

Embodiment 2

[0042] Embodiment 2 PI dye is to Streptomyces aureus spore staining:

[0043] After taking the sample and processing it, use sterile tweezers to place the slide into an EP tube filled with PBS (pH 7.4) buffer solution, shake it thoroughly for 1 min, and 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 a pure spore suspension of Streptomyces aureus. Dilute pure Streptomyces aureus spore suspension to a concentration of 10 with PBS 5 ~10 6 spores / mL. Mix an equal volume of Streptomyces aureus spore suspension with 10 μg / mL PI dye, and place in a 4°C refrigerator in the dark for 20 minutes for staining incubation.

[0044] Embodiment 3 Flow Cytometry Detection:

[0045] Detection should establish a control test to remove non-specific binding or background. A sample not labeled with any fluoresc...

Embodiment 4

[0046] Embodiment 4 aureomycin color reaction

[0047] According to the color reaction of aureomycin and samarium ions as the primary screening method. Chlortetracycline and samarium ions form light yellow complexes under near-neutral conditions, and have a maximum light absorption at 405nm. It is considered that after the fermentation is finished, the colored raw materials in the medium will have a certain impact on the measurement results. Use the production fermentation broth formula to cultivate and uncultivate bacteria in the orifice plate at the same time. The results show that the fermentation liquid of uncultivated cells also has a certain OD value, indicating that the color of the fermentation liquid itself does have an impact on the determination of the OD value, but the OD value of the fermentation liquid of the cultured cells is greater than that of the fermentation liquid of the uncultivated cells, so the fermentation liquid The color interference itself does no...

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Abstract

The invention discloses a method for high throughput screening of a chlortetracycline high-yield strain based on flow cytometry, and belongs to the technical field of high throughput screening. According to the method disclosed by the invention, by virtue of a PI streptomyces aureofaciens spore staining method, the activity of streptomyces aureofaciens spores is detected through flow cytometry, so that activity distinguishing and absolute counting of the streptomyces aureofaciens spores are achieved. A living spore group, obtained from analysis, is further sorted and cultivated, so that a mode is provided for the high throughput screening of the streptomyces aureofaciens. With the adoption of a solid-liquid combining high throughput cultivation scheme on the sorted living spores, the growth rate of a streptomyces aureofaciens pore plate is effectively improved. A chromogenic reaction and porous plate detection method, as a convenient and rapid detection means, is capable of simultaneously detecting a great amount of samples. The method, which sorts the active streptomyces aureofaciens based on the flow cytometry, provides a novel and effective mode for the high-throughput screening of the streptomyces aureofaciens high-yield strain.

Description

technical field [0001] The invention relates to a method for high-throughput screening of aureomycin high-yield strains based on flow cytometry, and belongs to the technical field of high-throughput screening. Background technique [0002] Chorotetracycline (CTC) is a broad-spectrum antibiotic belonging to the tetracycline class. Chlortetracycline can inhibit bacteria, promote growth, have high feed utilization rate and low residual amount in human body. Its production technology is relatively mature, and its production cost is relatively low. It has become the most widely used antibacterial and growth-promoting agent in the feed industry. At present, the productive rate of chlortetracycline in domestic factories is still low (titer is about 18000 μg / mL), so it is necessary to increase the productive rate of chlortetracycline. Aureomycin is a secondary metabolite obtained from the fermentation of Streptomyces aureofaciens. Its yield and synthesis traits are determined by m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/01C12N1/20C12R1/49
CPCC12N1/20C12N15/01
Inventor 周景文陈坚王得明陈玥张庆辉曹晓梅堵国成
Owner JIANGNAN UNIV
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