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Method for electroporation of Staphylococcus carnosus and its application

A technology of staphylococcus meatus and electrotransformation is applied in the field of electrotransformation of staphylococcus meatus, which can solve the problems of poor repeatability and low conversion rate, and achieve the effect of improving the electrotransformation efficiency

Inactive Publication Date: 2017-12-22
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Staphylococcus carnosus is a gram-positive bacterium, and the electroporation method is better than the protoplast transformation method, but there are still shortcomings such as low transformation rate and poor repeatability

Method used

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  • Method for electroporation of Staphylococcus carnosus and its application
  • Method for electroporation of Staphylococcus carnosus and its application
  • Method for electroporation of Staphylococcus carnosus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Optimization of Electrotransformation Conditions for Staphylococcus carnosus

[0081] 1. Plasmid extraction and quantitative detection

[0082] (1) Take 4 mL of the overnight bacterial culture, centrifuge at 12,000 rpm for 1 min, and discard the supernatant.

[0083] (2) Add 250 μL of solution (P1), use a pipette gun to blow and suck the bacterial pellet repeatedly to suspend it thoroughly.

[0084] (3) Add 250 μL solution II (P2), gently turn it up and down 6-8 times to fully lyse the bacteria, then add 350 μL solution III (P3), immediately turn it up and down gently 6-8 times, and centrifuge at 12000 rpm for 10 minutes.

[0085] (4) Add the supernatant collected in the previous step to the adsorption column, centrifuge at 12000rpm for 1min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0086] (5) Add 700 μL of rinse solution (PW), centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and pu...

Embodiment 2

[0108] Optimization of Electrotransformation Conditions for Staphylococcus carnosus

[0109] The difference between this example and Example 1 lies in that the selected shuttle vector is different, and pBT2-ET-4C-5R-EGFP is used in Example 2, and the rest of the experimental steps and conditions are the same.

[0110] 1. Plasmid extraction and quantitative detection

[0111] (1) Take 4 mL of the overnight bacterial culture, centrifuge at 12,000 rpm for 1 min, and discard the supernatant.

[0112] (2) Add 250 μL of solution (P1), use a pipette gun to blow and suck the bacterial pellet repeatedly to suspend it thoroughly.

[0113] (3) Add 250 μL solution II (P2), gently turn it up and down 6-8 times to fully lyse the bacteria, then add 350 μL solution III (P3), immediately turn it up and down gently 6-8 times, and centrifuge at 12000 rpm for 10 minutes.

[0114] (4) Add the supernatant collected in the previous step into the adsorption column, centrifuge at 12000rpm for 1mi...

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Abstract

The invention discloses a method for electroporation of Staphylococcus carnosus. The method comprises the following three steps: preparation of competent cells, electroporation, and resuscitation and culture. In the step of preparation of competent cells, Staphylococcus carnosus ATCC 51365 is activated in a LB medium and then inoculated into a growth medium B2, then undergoes shaking culture until OD578 is 0.6, and then is washed with a GS electric shock buffer. In the step of electroporation, to be transformed plasmids and the competent cells are uniformly mixed and subjected to ice-bath treatment and electroporation. In the step of resuscitation and culture, 1 mL of a LBM resuscitation medium is rapidly added into an electric shock cup after electric shock, the plasmids and the competent cells are taken out after uniform mixing and subjected to resuscitation and culture for 4 h, and the plasmids and the competent cells then coat a plate for culture overnight so as to obtain recombinant Staphylococcus carnosus with the introduced plasmids. With the method provided by the invention, conversion efficiency reaches 5.95 * 10<3> CFU / [mu]g DNA, so the conversion efficiency of Staphylococcus carnosus is greatly improved.

Description

technical field [0001] The invention relates to the technical field of bacterial transformation, in particular to an electrotransformation method of Staphylococcus carnosus, belonging to the field of biotechnology. Background technique [0002] Staphylococcus meatus ( Staphylococcus carnosus ) is a typical Gram-positive bacterium, and it is currently a food-grade GRAS (generally regarded as safe) strain that is fully confirmed in the Staphylococcus genus. ten years thing. Staphylococcus xylosus ( S. xylosus ) some strains have also been confirmed as GRAS strains, and the rest are pathogenic bacteria or conditional pathogenic bacteria, such as Staphylococcus aureus ( S. aureus ), Staphylococcus epidermidis ( S. epidermidis )Wait. Staphylococcus meatus ( S. carnosus ), as the key strain for forming the flavor of meat products in the meat product fermentation industry, it has long been used as the starting culture for the production of dry ham and dry sausage in E...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/64C12N15/74C12R1/44
CPCC12N15/64C12N15/74
Inventor 高强张变强唐巧巧朱燕
Owner TIANJIN UNIV OF SCI & TECH
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