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Transformation method of lactobacillus brevis

A technology of Lactobacillus brevis and electrotransformation, which is applied in the field of food microorganisms and can solve problems such as inapplicability

Active Publication Date: 2019-06-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as verified by the inventors, this electroporation method is not applicable to other Lactobacillus brevis

Method used

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  • Transformation method of lactobacillus brevis
  • Transformation method of lactobacillus brevis
  • Transformation method of lactobacillus brevis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Transformation of Lactobacillus brevis 2-34 with plasmid pNZ5319

[0034] (1) Electrotransformation of Lactobacillus brevis 2-34 with plasmid pNZ5319

[0035] 1. Preparation of Competent Cells of Lactobacillus brevis 2-34

[0036] Lactobacillus brevis 2-34 preserved in glycerol tubes was inoculated in MRS liquid medium at 2% inoculation amount, and cultured at 37°C for 12 hours; transferred to MRS liquid medium containing 1% glycine at 2% inoculum amount, Cultivate statically at 37°C for 24 hours; transfer to MRS liquid medium containing 1% glycine according to 2% inoculum size, and culture statically at 37°C until OD 600 0.8-1.0.

[0037] Put the culture solution in ice bath for 15min, centrifuge at 5000rpm, 4°C for 10min, and collect the bacterial cells; wash the bacterial cells with pre-cooled Washingsolution (WB) twice, and centrifuge at 6000rpm, 4°C for 10min, to collect the bacterial cells; Wash once with pre-cooled Electroporation Buffer (EB), centri...

Embodiment 2

[0049] Example 2 Transformation of Lactobacillus brevis ATCC 367 with plasmid pNZ5319

[0050] (1) Electrotransformation of Lactobacillus breve ATCC 367 with plasmid pNZ5319

[0051] Prepare the competent cells of Lactobacillus brevis ATCC 367 according to the method in Example 1, get 450ng of plasmid pNZ5913, add in 50 μ L of Lactobacillus brevis ATCC 367 competent cells, perform electroporation according to the steps of Example 1, recover after electroporation, The primers used for bacterial liquid coating, selection of positive transformants, identification of positive transformants, and PCR amplification identification are still the primer sequences in Table 1.

[0052] Positive transformants were identified as figure 2 As shown, the amplified product was identified by 1% agarose gel electrophoresis, and its size was 1000bp, which was consistent with the expected size. After gel cutting and recovery, it was analyzed by DNA sequencing, and its sequence was the same as the...

Embodiment 3

[0056] Example 3 Transformation of Lactobacillus brevis TMW 1.2112 with plasmid pNZ5319

[0057] (1) Electrotransformation of Lactobacillus brevis TMW 1.2112 with plasmid pNZ5319

[0058] Prepare the competent cells of Lactobacillus brevis TMW 1.2112 according to the method in Example 1, take 450ng of plasmid pNZ5913, add in 50 μ L Lactobacillus brevis TMW 1.2112 competent cells, perform electroporation according to the steps of Example 1, recover after electroporation, The primers used for bacterial liquid coating, selection of positive transformants, identification of positive transformants, and PCR amplification identification are still the primer sequences in Table 1.

[0059] Positive transformants were identified as image 3 As shown, the amplified product was identified by 1% agarose gel electrophoresis, and its size was 1000bp, which was consistent with the expected size. After gel cutting and recovery, it was analyzed by DNA sequencing, and its sequence was the same ...

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Abstract

The invention discloses a transformation method of lactobacillus brevis, and belongs to the technical field of food microorganisms. Since biochemical characteristics, molecular constitutions and immunological characteristics of bacterial strains of different species of lactic acid bacteria are all different, improvement is made on the basis of an existing electrotransformation method for lactococcus lactis and lactobacillus brevis which does not contain endogenous plasmids. The lactobacillus brevis is continuously activated twice separately through an MRS culture medium and an MRS culture medium containing 1% glycine, and continues to be cultured until the thalli reach a high concentration, then the lactobacillus brevis is collected, competent cells are prepared, and the concentration andactivity of the competent cells are improved accordingly. When exogenous DNA fragments are subjected to electrotransformation, the electric shock condition is 2.4 KV and 3.5 ms, and the electrotransformation efficiency is improved. As is checked, the competent cells are successfully transformed through exogenous genes, and the problems of preparation and transformation in the process of electrotransformation of wild lactobacillus brevis into the competent cells are solved.

Description

technical field [0001] The invention relates to a transformation method of Lactobacillus brevis, belonging to the technical field of food microorganisms. Background technique [0002] Lactic acid bacteria is a general term for a class of Gram-positive bacteria that produce by-products such as lactic acid, ethanol, and carbon dioxide through homofermentation or heterofermentation. They play an important role in the food industry such as wine, soy sauce, sausage, and other fermented foods and alcoholic beverages. Beneficial effects, such as adjusting the pH value, maintaining and improving the brewing micro-ecological environment, promoting saccharification and fermentation, and promoting the production of flavor substances and their precursors, etc. Among them, Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus fermentum are the dominant strains. However, lactic acid bacteria will also produce some toxic metabolites after fermentation, such as D-lactic acid, ni...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12R1/24
Inventor 李晓敏刘晓慧蔡国林吴殿辉陆健
Owner JIANGNAN UNIV
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