Method for improving acquisition efficiency of electroporated positive recombinants of escherichia coli

A technology of Escherichia coli and acquisition efficiency, applied in the field of bioengineering, can solve the problems of inaccurate screening and low electrotransformation efficiency of Escherichia coli, and achieve the effects of improving identification accuracy and improving electroconversion efficiency.

Inactive Publication Date: 2017-05-10
XI'AN UNIVERSITY OF ARCHITECTURE AND TECHNOLOGY
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Problems solved by technology

[0003] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for improving the efficiency of Escherichia coli electrotransformation positive recombinants, thereby solving the existing problems of low Escherichia coli electrotransformation efficiency and inaccurate screening

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  • Method for improving acquisition efficiency of electroporated positive recombinants of escherichia coli
  • Method for improving acquisition efficiency of electroporated positive recombinants of escherichia coli
  • Method for improving acquisition efficiency of electroporated positive recombinants of escherichia coli

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Embodiment 1

[0040] This example provides a method for improving the efficiency of Escherichia coli electrotransformation positive recombinants, including the preparation method of Escherichia coli competent cells and the acquisition method of exogenous linear target gene DNA fragments containing kanamycin resistance gene. The invention designs a 50bp homology arm sequence and constructs an exogenous DNA fragment containing a kanamycin resistance gene. Second: using Escherichia coli ATCC25922 as the recipient bacteria, prepare competent Escherichia coli E.coli / PKD46, in which the concentration of arabinose is 15mmol / L, and finally introduce the exogenous DNA fragment into Escherichia coli, and the electroporation voltage is 1.8kV.

[0041] The specific steps are as follows (taking the knockout of Escherichia coli ppk gene and recombining the foreign gene DNA fragment into Escherichia coli as an example):

[0042] 1. Construction of exogenous linear target DNA fragment containing kanamycin ...

Embodiment 2

[0072] This embodiment carries out screening verification to the result of embodiment 1

[0073] 1. Screening of recombinant bacteria

[0074] (a) After colonies grow on the nutrient agar plate containing kanamycin (25ug / ml), pick a single colony and inoculate it into 50ml LB medium. After culturing at 30°C and 170rpm for 5h, raise the temperature to 42°C and continue culturing at 170rpm for 10h;

[0075] (b) Using the streak separation method, take a loop of bacterial solution, inoculate them on nutrient agar plates containing kanamycin (25ug / ml) and ampicillin (60ug / ml) respectively, and culture overnight at 30°C;

[0076] (c) Screening and strain preservation Pick the colony that can grow on the plate containing kanamycin but not on the plate containing ampicillin, inoculate into LB medium containing kanamycin, overnight at 37°C After culturing for 16 hours, inoculate to nutrient agar slant medium at 30°C and store at 4°C for later use.

[0077] 2. Molecular identificati...

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Abstract

The invention provides a method for improving the acquisition efficiency of electroporated positive recombinants of escherichia coli. The method comprises the steps of (1) building an exogenous linear target DNA segment containing a kanamycin resistance gene; (2) preparing electroporation-competent cells of the escherichia coli; (3) carrying out gene integration on the exogenous linear target DNA segment and the electroporation-competent cells of the escherichia coli; and (4) screening positive recombinants of the escherichia coli, wherein identification primers for screening the positive recombinants of the escherichia coli include a forward primer and a reverse primer. Through the designed short-arm homologous sequence range, the exogenous DNA segment and bacterial chromosome are more easily exchanged and recombined. The electroporation efficiency can be improved through the provided recombinant electroporation voltage and arabinose concentration. The positive recombinants are further identified through the designed forward and reverse identification primers, and the identification accuracy is improved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for electrotransformation of microorganisms, in particular to a method for improving the efficiency of obtaining Escherichia coli electrotransformation positive recombinants. Background technique [0002] The application of electroporation technology in the biological field to obtain engineering bacteria with certain functions is a very practical technology. However, there are problems such as cumbersome transformation process and low recombination efficiency in the transformation process, and the problem of inaccurate screening results in recombinant screening. There are many factors that affect the recombination rate. The length of the homology arm, the concentration and time of arabinose induction, the recovery time, temperature, and the transformation voltage will all affect the transformation efficiency, and the most important factors are the length of the homo...

Claims

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Application Information

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IPC IPC(8): C12N15/90
CPCC12N15/902
Inventor 南亚萍周国标袁林江
Owner XI'AN UNIVERSITY OF ARCHITECTURE AND TECHNOLOGY
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