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Preparation and transformation method of streptococcus thermophilus competent cells

A technology of competent cells and Streptococcus thermophilus, applied in the biological field, can solve the problems of low transformation efficiency and achieve remarkable technological progress, high transformation efficiency and simple operation

Inactive Publication Date: 2018-07-06
UNIV OF SHANGHAI FOR SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the above technical problems in the prior art, the invention provides a method for preparing and transforming competent cells of Streptococcus thermophilus. The method for preparing and transforming competent cells for Streptococcus thermophilus should solve the existing The technical problem of low conversion efficiency of Streptococcus thermophilus electrotransformation in technology

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  • Preparation and transformation method of streptococcus thermophilus competent cells
  • Preparation and transformation method of streptococcus thermophilus competent cells
  • Preparation and transformation method of streptococcus thermophilus competent cells

Examples

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Embodiment 1

[0039]Example 1: Establishment of Competent Preparation Method for Streptococcus thermophilus AR479

[0040] According to the research on the electrotransformation method of lactic acid bacteria reported in the literature, the following five commonly used competent preparation methods were first tried:

[0041] Method 1: Streak activation of glycerol-preserved strain AR479 at -80°C, culture LM17 overnight, inoculate 1% inoculum into LM17 containing threonine (100mM), culture at 37°C until OD 600 0.6-0.8, centrifuge at 5000x g at 4°C for 10 minutes. Wash twice with washing buffer (0.5M sucrose, 10% glycerol), then resuspend with resuspension buffer (10% glycerol and 30% PGE2000 solution), 100 μL per tube, store at -80°C, and use electrotransformation for later use.

[0042] Method 2: LM17 liquid culture overnight, inoculate LM17 containing lysozyme (10 mg / mL) with 1% inoculum, and culture to OD at 37°C 600 0.6-0.8, centrifuge at 5000x g at 4°C for 10 minutes. Wash twice wi...

Embodiment 2

[0054] Example 2: Optimization of Electrotransformation Conditions for Streptococcus thermophilus AR479

[0055] In the electroconversion process, the electric field strength is a key factor. Electroporation is the use of instantaneous high-voltage electric shock to the cell membrane to form holes so that DNA molecules can enter the cell. During the electric shock process, the voltage is too low or too high to make foreign DNA molecules enter the cells effectively. However, within a certain range of electric field strength, the stronger the electric field strength, the more holes will be produced on the cell membrane, the better the permeability, and the easier it is for foreign DNA molecules to enter the cell. The electroconversion efficiency of AR479 increases with the increase of electric field intensity ( figure 2 ), when the electric field strength reaches 12.5kV / cm, the electric conversion efficiency reaches the highest, which is 6×10 5 CFU / μg DNA. With the further ...

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Abstract

The invention provides a preparation and transformation method of streptococcus thermophilus competent cells. The preparation and transformation method of the streptococcus thermophilus competent cells comprises the following steps: cultivating the streptococcus thermophilus in an LM17 culture medium until OD600 is 0.3 to 0.9, mixing the LM17 culture medium and an SLM17 culture medium according tothe ratio of 1:1, performing incubation at 37 DEG C for 1 hour, centrifugally connecting thallus at 4 DEG C, washing cells with a precooled buffer solution to obtain competent cells, mixing the competent cells and pIB184 plasmid uniformly, performing electric shock by using electric field intensity of 12.5 kV / cm, and performing resuscitation in an LM17MC2 culture medium for 3 to 8 hours to obtainthe highest electric transformation efficiency, which is 1.3*106 CFU / ug DNA, of the streptococcus thermophilus. The method provided by the invention is simple in operation and high in transformationefficiency; and a new technical method is provided for genetic modification of the streptococcus thermophilus and molecular mechanism for illuminating phenotypic characteristic.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a streptococcus thermophilus, in particular to a method for preparing and transforming competent cells of the streptococcus thermophilus. Background technique [0002] At present, the transformation methods of lactic acid bacteria are mainly divided into two types: one is the protoplast method. Due to the fragility of the protoplasts, it is difficult and very cumbersome to prepare the protoplasts of Streptococcus thermophilus, and polyethylene glycol is also required to promote DNA entry. The second is the electroconversion method, which has been widely used because of its simple operation and high conversion efficiency. Electroporation is currently the most effective transformation method for lactic acid bacteria such as Streptococcus thermophilus, and efficient transformation of Streptococcus thermophilus is the prerequisite for heterologous expression of exogenous gene products in S...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N13/00C12R1/01
CPCC12N1/20C12N13/00
Inventor 熊智强艾连中孔令慧王光强夏永军张汇
Owner UNIV OF SHANGHAI FOR SCI & TECH
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