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Electroporation genetic transformation method of lactobacillus plantarum

A genetic transformation method, Lactobacillus plantarum technology, applied in microorganism-based methods, biochemical equipment and methods, and introduction of foreign genetic material using vectors, etc., can solve the problems of complicated operation, unfavorable wide application, low transformation efficiency, etc. The effect of increased ability

Inactive Publication Date: 2014-05-28
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The former is complicated to operate and has low transformation efficiency, which is not conducive to wide application, especially for strains with industrial application value, while the latter is relatively simple to operate and relatively high transformation efficiency, so the electroporation method is mainly used today

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Genetic transformation of exogenous erythromycin-resistant gene to Lactobacillus plantarum by electroporation

[0029] Using the method described in the Summary of the Invention, the foreign plasmid pMG36e containing the erythromycin resistance gene was introduced into Lactobacillus plantarum, and the stably transformed engineering bacteria were obtained by screening with erythromycin, and a higher transformation efficiency was obtained. The specific method is as follows:

[0030] 1. Preparation of Lactobacillus plantarum competent cells:

[0031] 1) Activation of Lactobacillus plantarum strains is carried out on the MRS medium plate, and the culture temperature is 37°C. The final concentration of MRS solid medium is: peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, anhydrous glucose 20.0g, dipotassium hydrogen phosphate 2.0g, sodium acetate 5.0g, magnesium sulfate 0.2g, manganese sulfate 0.05g, Ammonium citrate 2.0g, Tween 1.0g, 2% agar powder, add 1...

Embodiment 2

[0047] Example 2: Genetic transformation of exogenous erythromycin-resistant gene to Lactobacillus plantarum by electroporation

[0048] Using the method described in the Summary of the Invention, the foreign plasmid pMG36e containing the erythromycin resistance gene was introduced into Lactobacillus plantarum, and the stably transformed engineering bacteria were obtained by screening with erythromycin, and a higher transformation efficiency was obtained. The specific method is as follows:

[0049] 1. Preparation of Lactobacillus plantarum competent cells:

[0050] 1) Activation of Lactobacillus plantarum strains is carried out on the MRS medium plate, and the culture temperature is 37°C. The final concentration of MRS solid medium is: peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, anhydrous glucose 20.0g, dipotassium hydrogen phosphate 2.0g, sodium acetate 5.0g, magnesium sulfate 0.2g, manganese sulfate 0.05g, Ammonium citrate 2.0g, Tween 1.0g, 2% agar powder, add 1...

Embodiment 3

[0066] Example 3: Genetic transformation of exogenous erythromycin-resistant gene to Lactobacillus plantarum by electroporation

[0067] Using the method described in the Summary of the Invention, the foreign plasmid pMG36e containing the erythromycin resistance gene was introduced into Lactobacillus plantarum, and the stably transformed engineering bacteria were obtained by screening with erythromycin, and a higher transformation efficiency was obtained. The specific method is as follows:

[0068] 1. Preparation of Lactobacillus plantarum competent cells:

[0069] 1) Activation of Lactobacillus plantarum strains is carried out on the MRS medium plate, and the culture temperature is 37°C. The final concentration of MRS solid medium is: peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, anhydrous glucose 20.0g, dipotassium hydrogen phosphate 2.0g, sodium acetate 5.0g, magnesium sulfate 0.2g, manganese sulfate 0.05g, Ammonium citrate 2.0g, Tween 1.0g, 2% agar powder, add 1...

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Abstract

The invention provides an efficient genetic transformation method of exogenous gene of lactobacillus plantarum by using an optimized electroporation technology. Exogenous DNA (Desoxvribose Nucleic Acid) enters the lactobacillus plantarum through an efficient electroporation method of adding exogenous gene (plasmid) at an optimal concentration and under a bacteria optimum growth condition, restoring a culture medium to prepare a sucrose solution at optimal concentration and controlling a recovery time after electrically shocking. The efficient genetic transformation method of the exogenous gene of the lactobacillus plantarum has the benefits that the efficient genetic transformation method of the exogenous gene of the lactobacillus plantarum can be applied to molecular biology study of the lactobacillus plantarum, for example, the study of a molecular mechanism of the lactobacillus plantarum for reducing cholesterol and a molecular mechanism of the lactobacillus plantarum for producing acid and resisting cholate; the efficient genetic transformation method of the exogenous gene of the lactobacillus plantarum also can be used for carrying out genetic engineering on the lactobacillus plantarum so as to carry out genetic improvement and improve the capability of reducing cholesterol.

Description

(1) Technical field [0001] The invention relates to the electroporation genetic transformation technology of plant lactobacillus, which is suitable for the fields of genetic engineering of plant lactobacillus, molecular biology research of plant lactobacillus and the like. (2) Background technology [0002] As one of the normal intestinal flora of humans and animals, lactic acid bacteria have been proven to have many beneficial functions, and are generally regarded as safe and non-toxic (Generally regarded as safe, GRAS). Due to the important application of lactic acid bacteria in fermentation, agriculture, food, bioprocessing and even medicine, research on the genetics of lactic acid bacteria has been carried out abroad as early as the 1980s. The research on the genetic system of lactic acid bacteria is of great significance for analyzing the important industrial characteristics of lactic acid bacteria and transforming them through genetic, metabolic and protein engineering...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12R1/25
Inventor 白小佳张新利王艳萍王金菊
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY