Method for high throughput screening of chlortetracycline high-yield strains

A technology of high-yield strains and screening methods, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of long detection time, cumbersome detection methods, and long cultivation cycle, etc., to prevent the accuracy of screening Insufficient, increased spore growth speed, increased efficiency and accuracy effects

Inactive Publication Date: 2016-12-07
JIANGNAN UNIV
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Problems solved by technology

[0003] The screening of existing aureomycin strains is generally detected by HPLC method after shake flask fer...

Method used

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  • Method for high throughput screening of chlortetracycline high-yield strains

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Embodiment 1

[0044] Embodiment 1: optimal plate culture medium selection

[0045] Five different formulations of slant plate medium were prepared, and the diluted sterile Streptomyces aureus suspension was spread on the solid slant medium. Observe the growth of Streptomyces aureus on the plate medium, and detect its resistance production. It was found that the strain on the solid plate medium No. 1 could grow rapidly, and the spores grew well, and the color of the plate medium would not interfere with the subsequent selection of bacteria by the monoclonal selector. For other media, either the strains grow slowly, or the color of the plate media interferes with the subsequent selection of bacteria by the monoclonal picker.

Embodiment 2

[0046] Example 2: ARTP mutagenesis

[0047] The slant strain of the starting strain was cultured at 34°C for 4-6 days, scraped an appropriate amount of spores and dispersed them in a test tube with glass beads containing 2mL of normal saline, took 10μL on a sterilized special slide, and put it in the ARTP mutagenesis instrument On the stage, adjust the power to 10-100w, and irradiate for 0, 10, 20, 30, 40, 50, 60s respectively. Use tweezers to pick up the irradiated slides into the EP tube containing physiological saline, and shake After fully shaking on the instrument, gradient dilution was performed. Pipette 100 μL of the diluted solution and spread it evenly on the plate. After culturing in a constant temperature incubator at 34°C for 4 days, count the number of colony-forming units on each plate, and then calculate the lethal rate for each mutagenesis treatment time. Under the condition of incident power of 10-100W and helium flow rate of 1-10SLM, the irradiation time of...

Embodiment 3

[0048] Embodiment 3: aureomycin color reaction

[0049] 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 itself has little effect o...

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Abstract

The invention discloses a method for high throughput screening of chlortetracycline high-yield strains and belongs to the technical field of metabolic engineering. Through a comparison of growth and yield of streptomyces aureofaciens on five flat plate culture mediums, an optimal culture medium is selected, and guarantee is provided for starting strains. Through a comparison of two primary screening methods which are color reaction between chlortetracycline and Sm3+ and a colorimetric method of co-heat of chlortetracycline and hydrochloric acid, the simple and accurate color reaction is selected as the primary screening method. Repeatability and accuracy of nitric acid color reaction serving as a primary screening means are verified.

Description

technical field [0001] The invention relates to a method for screening high-yield aureomycin mutagenic strains, belonging to the technical field of microbial production. 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 multiple genes and its metabolic network ...

Claims

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Application Information

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IPC IPC(8): C12N15/01C12R1/49
CPCC12N15/01
Inventor 周景文陈坚曹晓梅堵国成曾伟主
Owner JIANGNAN UNIV
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