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Method for high-flux flat plate primary screening of strains with high 4GT (4-Alpha-Glycosyltransferase) activity

A high-throughput, high-yield strain technology, applied in the field of bioengineering, can solve problems such as heavy workload, cumbersome operations, and large human errors, and achieve the effects of improving efficiency, increasing the number of colonies, and reducing the intensity of screening work.

Inactive Publication Date: 2019-12-31
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, if the traditional breeding method without high-throughput screening is used to select a large number of mutant libraries, it needs to go through multiple primary screenings and secondary screenings, which is cumbersome to operate, with a large workload, and human error is also large.

Method used

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  • Method for high-flux flat plate primary screening of strains with high 4GT (4-Alpha-Glycosyltransferase) activity
  • Method for high-flux flat plate primary screening of strains with high 4GT (4-Alpha-Glycosyltransferase) activity
  • Method for high-flux flat plate primary screening of strains with high 4GT (4-Alpha-Glycosyltransferase) activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Plate primary screening secreting 4-alpha-glycosyltransferase strain

[0036] Cultivation of strains in liquid medium: Inoculate activated strain pET32a-4GT / BL21(DE3), strain pET32a / BL21(DE3) and Escherichia coli BL(DE3) in 30 mL of LB liquid with 1% inoculum in the culture base. Among them, 100 μg / mL ampicillin antibiotic was added to the medium containing strain pET32a-4GT / BL21(DE3) and strain pET32a / BL21(DE3), while no antibiotic was added to the culture medium of Escherichia coli BL21(DE3), and at 37 Cultivate on a shaker at 200 rpm. OD of bacteria solution 600 When the value increased to 0.6, 1 mM inducer IPTG was added, and the fermentation was carried out for 12 hours. After the fermentation is completed, the fermentation products of each bacteria are centrifuged separately. With the aid of iodine chromogenic solution, the enzyme activity chromogenic reaction was carried out on the fermentation supernatant of each strain. As can be seen from the re...

Embodiment 2

[0038] Example 2 Plate primary screening with different substrates secreting 4-alpha-glycosyltransferase strains

[0039] Cultivation of bacterial strains on solid media: LB solid media containing 1% (w / v) sucrose, maltose, soluble starch, corn starch, wheat starch and potato starch were prepared respectively. Wherein, the medium contains 100 μg / mL ampicillin and 1 mM IPTG. The activated strains pET32a / BL21(DE3), pET32a-4GT / BL21(DE3) and pET32a(ΔTrx)-4GT / BL21(DE3) were inoculated onto the LB solid medium plates of the above different substrates. After each plate was placed upside down in an incubator at 37° C. for 24 hours, the plate was covered with iodine color development solution, and the color development results around each single colony were observed.

[0040] It can be seen from the results that on the LB solid medium plates of the sucrose group and the maltose group, the iodine color reaction was colorless; on the soluble starch plate, the iodine color reaction was r...

Embodiment 3

[0041] Example 3 Plate preliminary screening with different concentrations of substrates secreting 4-alpha-glycosyltransferase strains

[0042] LB solid medium containing 1%, 3% and 5% (w / v) potato starch were respectively configured, containing 100 μg / mL ampicillin antibiotic and 1 mM IPTG in the medium, and the activated bacterial strains pET32a / BL21(DE3), pET32a-4GT / BL21(DE3) and pET32a(ΔTrx)-4GT / BL21(DE3) were inoculated on LB solid medium plates with different concentrations of potato starch as above. After each plate was placed upside down in an incubator at 37° C. for 24 hours, the plate was covered with iodine color development solution, and the color development results around each single colony were observed.

[0043] It can be seen from the results that, as the concentration of potato starch increases, in the control group, there is no purple circle around the single colony pET32a / BL21(DE3) (No. 1), which is consistent with the expected phenomenon. In the experimen...

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Abstract

The invention discloses an efficient high-flux screening method of 4GT (4-Alpha-Glycosyltransferase) high-producing strains based on combination of a cell microporous plate and a screening flat plateformed by a specific culture medium, and belongs to the technical field of bioengineering. The method comprises the steps of building a high-flux flat plate primary screening method; optimizing the high-flux flat plate primary screening method; performing sequentialermr-PronePCR mutagenesis on 4GT genes; performing high-flux flat plate primary screening based on the cell microporous plate on the strains; and performing shake flask re-screening on the strains. A conventional screening method is improved; the work intensity is effectively reduced; and the screening efficiency of the high-producing strains is improved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a rapid and high-throughput method for breeding bacterial strains with high 4-alpha-glycosyltransferase activity based on selective plates and microporous cell culture plates. Background technique [0002] Starch is the main basic component of many staple foods such as rice, steamed buns, and noodles. Its textural properties are crucial to the quality and taste of foods. It is one of the main methods to optimize the physical and chemical properties of starch through food enzymes in order to improve the quality of starch-based food. The starch modified by 4-α-glycosyltransferase (abbreviated as 4GT) has improved retrogradation properties, improved digestion resistance, increased solubility, and has thermoreversible gel properties. These properties suggest that 4GT has great application prospects in the fields of food, cosmetics and medicine. [0003] At present, domestic r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48C12Q1/04C12N15/70
CPCC12Q1/48C12Q1/04C12N15/70G01N2333/91091G01N2333/245
Inventor 欧阳小英黄静杨婷朱锐万惠惠施瑞
Owner EAST CHINA NORMAL UNIV
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