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Electroporation method for streptococcus mutans

A technology of Streptococcus mutans, electroshock transformation, applied in biochemical equipment and methods, microorganism-based methods, other methods of inserting foreign genetic materials, etc., can solve the problems of bacterial cell death, poor transformation repeatability, etc., to overcome Toxicity, enhanced electroshock conversion efficiency and reproducibility

Inactive Publication Date: 2008-06-25
SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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Problems solved by technology

[0004] In the study on electric shock transformation of Streptococcus mutans, researchers found that culturing Streptococcus mutans in a glycine-containing medium for electric shock transformation would cause a large number of bacterial cells to die, which made the reproducibility of transformation poor. Cytotoxicity of glycine itself

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  • Electroporation method for streptococcus mutans

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Embodiment 1

[0017] 1. Preparation of experimental strains, plasmids and reagents

[0018] Streptococcus mutans strain UA159 (purchased from the School of Stomatology, Wuhan University) was used in the experiment, and the ampicillin-resistant pGL3 basic plasmid (purchased from ThermoFisher Scientific, Inc., USA) was used as the exogenous DNA for transformation.

[0019] Electroporation buffer (EPB) was prepared as follows: 5mM potassium phosphate (potassium phosphate), 0.4M sorbitol (sorbitol), 10% glycerol (glycerol), adjust the pH to 4.5, store at 4°C, and pre-cool in an ice bath before use.

[0020] 2. Preparation of Streptococcus mutans Competent Cells

[0021] Pick a single colony and inoculate it into BHI medium, and incubate on a shaker at 37°C until the absorbance is OD 660nm =0.5, add a glycine solution with a final concentration of 10%, and continue to incubate at 37° C. for 1 hr. After the culture solution was pre-cooled in an ice bath for 15 minutes, it was centrifuged at 300...

Embodiment 2

[0025] Effect of Different Final Concentrations of Glycine on the Transformation Efficiency of Streptococcus mutans by Electroshock

[0026] Streptococcus mutans strain UA159 (purchased from the School of Stomatology, Wuhan University) was used in the experiment, and the ampicillin-resistant pGL3 basic plasmid (purchased from ThermoFisher Scientific, Inc., USA) was used as the exogenous DNA for transformation.

[0027] Electroporation buffer (EPB) was prepared as follows: 5mM potassium phosphate (potassium phosphate), 0.4M sorbitol (sorbitol), 10% glycerol (glycerol), adjust the pH to 4.5, store at 4°C, and pre-cool in an ice bath before use.

[0028] Preparation of Streptococcus mutans competent cells: Pick a single colony and inoculate it into BHI medium, and incubate on a shaker at 37°C until the absorbance is OD 660nm =0.5, add different concentrations of glycine solutions (0, 2%, 5%, 10%, 20%), and continue to incubate at 37° C. for 1 hr. After the culture solution was p...

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Abstract

The invention relates to an electroporation transformation method, in particular to an electroporation transformation method to streptococcus mutans. The electroporation transformation method of the invention comprises the following steps: firstly, a streptococcus mutans UA159 strain, exogenous DNAs and electroporation transformation buffers are provided; secondly, competent cells of streptococcus mutans are prepared; thirdly, the competent cells are transformed by electroporation, wherein, the streptococcus mutans in the second step is inoculated into a BHI culture medium and incubated in a shaking table under the condition of 37 DEG C to absorbance OD660nm is equal to 0.5, and then 10 percent glycine solution is added. The electroporation transformation method to the treptococcus mutans disclosed by the invention has the advantages of obvious reinforcement of electroporation transformation efficiency and repeatability of the streptococcus mutans. Moreover, the invention is an effective means for gene transformation research of the streptococcus mutans and can be used for researching a cariogenic mechanism of the streptococcus mutans.

Description

technical field [0001] The invention relates to an electric shock transformation method, in particular to an electric shock transformation method for Streptococcus mutans. Background technique [0002] Caries is one of the main diseases that endanger human oral health. During the occurrence and development of caries, Streptococcus mutans, a resident bacterium in the oral cavity, plays an important role due to its strong ability to produce acid and resist acid. As the main cariogenic bacteria, Streptococcus mutans has received extensive attention and research. It has become an important means to study the pathogenic mechanism of Streptococcus mutans by introducing exogenous DNA fragments into it through DNA recombination technology to study the pathogenicity-related genes of Streptococcus mutans. [0003] The common gene transformation of Streptococcus mutans is carried out through electroporation transformation scheme, that is, the use of pulsed electric field to cause chan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87C12N1/21C12R1/46C12N15/74
Inventor 黄正蔚刘正
Owner SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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