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

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Problems solved by technology

However, as verified by the inventors, this electroporati

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

Examples

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Example Embodiment

[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 Lactobacillus brevis 2-34 competent cells

[0036] Inoculate the Lactobacillus brevis 2-34 preserved in the glycerol tube into MRS liquid medium at a 2% inoculum amount, and incubate at 37°C for 12 hours; transfer 2% inoculum to MRS liquid medium containing 1% glycine, Incubate at 37°C for 24 hours; transfer to MRS liquid medium containing 1% glycine according to 2% inoculum, and incubate at 37°C to OD 600 Is 0.8-1.0.

[0037] The culture solution was ice-bathed for 15 minutes, and centrifuged at 5000rpm for 10min at 4℃ to collect the bacteria; the bacteria cells were washed twice with pre-cooled Washingsolution (WB), and centrifuged at 6000rpm for 10min at 4℃ to collect the bacteria; for bacteria cells Wash once in pre-cooled Electroporation Buffer (EB), centrifuge at 8000 rpm, 4°C for 10 min...

Example Embodiment

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

[0050] (1) Plasmid pNZ5319 electrotransforms Lactobacillus brevis ATCC 367

[0051] Prepare competent cells of Lactobacillus brevis ATCC 367 according to the method in Example 1, take 450 ng of plasmid pNZ5913, add 50 μL of Lactobacillus brevis ATCC 367 competent cells, and perform electrotransformation according to the steps in Example 1, and recover after electrotransformation. Bacterial solution coating, selection of positive transformants and identification of positive transformants, and the primers used in PCR amplification identification are still the primer sequences in Table 1.

[0052] The positive transformant identification results are as follows figure 2 As shown, the amplified products were identified by 1% agarose gel electrophoresis, and their size was 1000 bp, which was consistent with the expected size. After the gel was cut and recovered, DNA sequencing analysis showed that the ...

Example Embodiment

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

[0057] (1) Plasmid pNZ5319 electrotransforms Lactobacillus brevis TMW 1.2112

[0058] Prepare competent cells of Lactobacillus brevis TMW 1.2112 according to the method in Example 1. Take 450 ng of plasmid pNZ5913 and add 50 μL of Lactobacillus brevis TMW 1.2112 competent cells. Follow the steps in Example 1 for electrotransformation, and recovery after electrotransformation. Bacterial solution coating, selection of positive transformants and identification of positive transformants, and the primers used in PCR amplification identification are still the primer sequences in Table 1.

[0059] The positive transformant identification results are as follows image 3 As shown, the amplified products were identified by 1% agarose gel electrophoresis, and their size was 1000 bp, which was consistent with the expected size. After the gel was cut and recovered, DNA sequencing analysis showed that the seq...

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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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