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Conversion method for introducing shuttle plasmids into brevibacterium flavum (or corynebacterium glutamicum)

A technology of Corynebacterium glutamicum and Brevibacterium flavum, applied in the field of wild type and auxotrophic type, can solve the problems of expensive medium raw materials and low transformation efficiency, and achieve the effects of clear thinking, good transformation effect and broad prospects.

Inactive Publication Date: 2014-08-06
SHANGHAI KING SHINE BIOLOGY TECH +1
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Problems solved by technology

Due to the characteristics of Brevibacterium flavum (or Corynebacterium glutamicum), it is difficult for competent cells prepared by general chemical methods to introduce plasmids into Brevibacterium flavum (or Corynebacterium glutamicum) cells, or commonly used in the construction of genetically engineered bacteria The transformation method is that the electric shock transformation is relatively easy to introduce the plasmid into the cells, but the raw materials of the culture medium prepared by the competent cells used for the electric shock transformation of Brevibacterium flavum (or Corynebacterium glutamicum) are relatively expensive, and the operation is relatively simple, but the transformation efficiency is not high. Voltage (setting of capacitance and resistance) and time parameters are the key parameters

Method used

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  • Conversion method for introducing shuttle plasmids into brevibacterium flavum (or corynebacterium glutamicum)
  • Conversion method for introducing shuttle plasmids into brevibacterium flavum (or corynebacterium glutamicum)
  • Conversion method for introducing shuttle plasmids into brevibacterium flavum (or corynebacterium glutamicum)

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preparation example Construction

[0027] The preparation method of LB solid medium (pH 7.2): Mix 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar powder and double distilled water thoroughly and dilute to 1 L with double distilled water; 121 High temperature sterilization for 20 min.

[0028]Preparation method of 10×inorganic salt mother liquor (PH 7.0): Dissolve 10 g of ammonium sulfate, 60 g of dipotassium hydrogen phosphate, 10 g of trisodium citrate and 2 g of magnesium sulfate heptahydrate in double distilled water and dilute to 1 L, 121 High temperature sterilization at ℃ for 20 min.

[0029] Preparation method of 20% glucose solution: Dissolve 20g of glucose in double-distilled water to 100 ml, filter and sterilize through a 0.22um bacterial filter, and store in a refrigerator at 4°C.

[0030] The preparation method of 2% casein hydrolyzate solution: 2g of casein hydrolyzate was dissolved in double distilled water and the volume was adjusted to 100ml, and sterilized by filtration with ...

Embodiment 1

[0031] Example 1 Transformation method of shuttle plasmid into wild-type Corynebacterium glutamicum ATCC 13032

[0032] Preparation of growth medium and induction medium

[0033] The preparation method of growth medium (1L): mix 10×inorganic salt mother liquor, 20% glucose solution, 2% casein hydrolyzate solution and double distilled water to obtain growth medium GM I; the solvent of growth medium is double distilled Water, solutes and their concentrations are as follows: ammonium sulfate 2 g / L, dipotassium hydrogen phosphate 14.8 g / L, trisodium citrate 1.9 g / L, magnesium sulfate 0.098 g / L, glucose 5 g / L, casein hydrolyzate 0.2 g / L.

[0034] Preparation method of induction medium (1L): mix 10× inorganic salt stock solution, 0.5 ml 0.1M CaCl 2 , 1ml 25 mM MgCl 2 , 20% glucose solution and double distilled water were mixed to obtain the induction medium GM II; the solvent of the induction medium was double distilled water, and the solute and its concentration were as follows:...

Embodiment 2

[0042] Example 2 Transformation method for introducing shuttle plasmid into auxotrophic Brevibacterium flavum AN78

[0043] Preparation of Growth Medium GM I and Induction Medium GM II

[0044] The preparation method of growth medium (1 L) GMI: mix 10X inorganic salt mother liquor, 20% glucose solution, 2% casein hydrolyzate solution and double distilled water to obtain growth medium; the solvent of growth medium is double distilled Water, solutes and their concentrations are as follows: ammonium sulfate 2 g / L, dipotassium hydrogen phosphate 14.8 g / L, trisodium citrate 1.9 g / L, magnesium sulfate 0.098 g / L, glucose 5 g / L, casein hydrolyzate 0.2 g / L, 0.25% amino acid mixture (His, Met, Pro).

[0045] The preparation method of induction medium (1 L) GM II: mix 10×inorganic salt mother solution, 20% glucose solution and double distilled water to obtain induction medium; the solvent of growth medium is double distilled water, and the solute and its concentration are as follows : ...

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Abstract

The invention discloses a rapid and effective method for converting recombinant plasmids that can perform shuttle expression in escherichia coli and brevibacterium flavum (or corynebacterium glutamicum) into corynebacterium glutamicum and auxotroph brevibacterium flavum. The method comprises two steps namely competent cell preparation and high-temperature culture and conversion: (1) inoculating auxotroph brevibacterium flavum (or corynebacterium glutamicum) into a specially-prepared culture medium to enable the auxotroph brevibacterium flavum (or corynebacterium glutamicum) to carry out rapid proliferation, and then transforming the auxotroph brevibacterium flavum (or corynebacterium glutamicum) to a culture medium with inefficient nutrients to prepare competent cells; (2) evenly mixing plasmids to be converted and the prepared competent cells, placing the mixture into an ice bath, culturing under a constant temperature, and finally introducing the plasmids into brevibacterium flavum (or corynebacterium glutamicum) to be converted. The method provided by the invention can reduce the restrictive enzyme digestion effect of brevibacterium flavum on shuttle plasmids, the shuttle plasmids can be stably copied and transmitted, and thus the method has the advantages of good conversion effect and convenient operation.

Description

technical field [0001] The invention relates to the field of bacterial transformation, in particular to a method for transforming and introducing plasmids into Brevibacterium flavum (or Corynebacterium glutamicum), including wild type and auxotrophic type. Background technique [0002] Brevibacterium flavum (or Corynebacterium glutamicum) is a Gram-positive bacterium widely found in nature. Due to the characteristics of Brevibacterium flavum (or Corynebacterium glutamicum), it is difficult for competent cells prepared by general chemical methods to introduce plasmids into Brevibacterium flavum (or Corynebacterium glutamicum) cells, or commonly used in the construction of genetically engineered bacteria The transformation method is that the electric shock transformation is relatively easy to introduce the plasmid into the cells, but the raw materials of the culture medium prepared by the competent cells used for the electric shock transformation of Brevibacterium flavum (or C...

Claims

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Application Information

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IPC IPC(8): C12N15/77C12N1/21C12R1/15C12R1/13
Inventor 王翠平马承国王开成张传军
Owner SHANGHAI KING SHINE BIOLOGY TECH
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