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Method for purifying propionibacterium strain plasmid

A technology of Propionibacterium and strains, which is applied in the field of plasmid purification of Propionibacterium strains, can solve the problems of plasmid screening, extraction and purification methods that are not mentioned, and achieve the effect of improving purity and extraction efficiency

Active Publication Date: 2012-01-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There were few reports on the research of Propionibacterium. P.Kiatpapan and Y.Murooka reported the construction of a vector suitable for Propionibacterium strains, but did not mention the specific plasmid screening, extraction and purification methods

Method used

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  • Method for purifying propionibacterium strain plasmid
  • Method for purifying propionibacterium strain plasmid
  • Method for purifying propionibacterium strain plasmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] (1) Inoculate 6 bottles of medium, 200mL per bottle, and culture at 30°C for 35h, centrifuge at 4°C, 10,000g for 3min, remove the supernatant, and obtain six tubes of bacterial sediment, numbered 1-6 respectively.

[0017] (2) Wash the six tubes of cells with 10 mL SET eluent, centrifuge at 4°C and 10,000 g for 3 min, remove the supernatant, and dry the pellet to obtain the wet weight.

[0018] (3) Add 10 mL of solution I to suspend thoroughly.

[0019] (4) Add 1mL lysozyme solution respectively, 37°C water bath, No. 1 water bath for 0 min, No. 2 water bath for 30 min, No. 3 water bath for 0 min, No. 4 water bath for 30 min, No. 5 water bath for 30 min, and No. 6 water bath for 60 min.

[0020] (5) Add 400mL proteinase K solution, 56°C water bath, No. 1 water bath for 0 min, No. 2 water bath for 0 min, No. 3 water bath for 30 min, No. 4 water bath for 30 min, No. 5 water bath for 60 min, and No. 6 water bath for 30 min;

[0021] (6) Add 20mL of newly prepared solution ...

Embodiment 2

[0030] (1) Inoculate 6 bottles of culture medium, incubate 300mL of each bottle at 30°C for 35 hours, centrifuge at 4°C and 10,000g for 3min, remove the supernatant, and obtain six tubes of bacterial sediment, numbered 1-6.

[0031] (2) Wash the six tubes of cells with 10 mL SET eluent, centrifuge at 4°C and 10,000 g for 3 min, remove the supernatant, and dry the pellet to obtain the wet weight.

[0032] (3) Add 30mL solution I to suspend thoroughly.

[0033] (4) Add 1mL mutanolysin solution respectively, 37°C water bath, No. 1 water bath for 0 min, No. 2 water bath for 30 min, No. 3 water bath for 0 min, No. 4 water bath for 30 min, No. 5 water bath for 30 min, and No. 6 water bath for 60 min.

[0034] (5) Add 300mL proteinase K solution, 56°C water bath, No. 1 water bath for 0 min, No. 2 water bath for 0 min, No. 3 water bath for 30 min, No. 4 water bath for 30 min, No. 5 water bath for 60 min, and No. 6 water bath for 30 min;

[0035] (6) Add 15mL of newly prepared solutio...

Embodiment 3

[0044] (1) Inoculate 6 bottles of culture medium, incubate 300mL of each bottle at 30°C for 35 hours, centrifuge at 4°C and 10,000g for 3min, remove the supernatant, and obtain six tubes of bacterial sediment, numbered 1-6.

[0045](2) Wash the six tubes of bacterial cells with 30 mL SET eluent, centrifuge at 4°C and 10,000 g for 3 min, remove the supernatant, and dry the precipitate to obtain the wet weight.

[0046] (3) Add 10 mL of solution I to suspend thoroughly.

[0047] (4) Add 1mL lysozyme solution and 1mL mutanolysin solution, respectively, in 37°C water bath, No. 1 water bath for 0 min, No. 2 water bath for 30 min, No. 3 water bath for 0 min, No. 4 water bath for 30 min, No. 5 water bath for 30 min, and No. 6 water bath for 60 min.

[0048] (5) Add 900mL proteinase K solution, 56°C water bath, No. 1 water bath for 0 min, No. 2 water bath for 0 min, No. 3 water bath for 30 min, No. 4 water bath for 30 min, No. 5 water bath for 60 min, and No. 6 water bath for 30 min; ...

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Abstract

The invention discloses a method for purifying propionibacterium strain plasmid. By performing pretreatment and subsequent treatment on thallus, high-purity plasmid can be obtained from a propionibacterium strain. Compared with the conventional method, the method has the advantages of greatly improving the concentration and the purity of the plasmid. The method lays a solid foundation for research in the aspect of molecular operation of propionibacterium and possibly has universal application significance to other gram-positive bacteria (such as lactic acid bacteria).

Description

technical field [0001] The invention relates to a method for purifying a plasmid, in particular to a method for purifying a plasmid of a Propionibacterium strain. Background technique [0002] Propionibacterium is a kind of Gram-positive, non-motile, non-spore-forming, anaerobic, oxygen-tolerant, and catalase-positive rod-shaped bacteria, and the cell wall is more complex than ordinary Gram-positive bacteria, and the combination is tighter . Propionibacterium is an important class of microorganisms for industrial use and has been widely used in the production of microorganisms B 12 , tetrapyrrole compounds and propionic acid, and are also used in the production of bread, cheese, and pharmaceuticals. Especially propionic acid, as a primary metabolite, is widely used in cellulose films, herbicides, and perfumes. Propionic acid is also a bacteriostatic agent, which can be used for food and grain preservation. [0003] Typical propionibacteria are a class of non-pathogenic b...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 陈坚堵国成刘龙诸葛鑫
Owner JIANGNAN UNIV
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