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Genetic recombination method of klebsiella pneumoniae by utilizing short homologous sequence

A pneumococcus, homologous sequence technology, applied in the field of gene recombination, to achieve the effect of simple operation

Active Publication Date: 2014-01-08
SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0007] However, for gene recombination of Klebsiella pneumoniae, existing technologies use longer homology arms, and the target gene needs to be cloned to construct long homology arms. A simpler and more convenient method is needed to promote research.

Method used

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  • Genetic recombination method of klebsiella pneumoniae by utilizing short homologous sequence
  • Genetic recombination method of klebsiella pneumoniae by utilizing short homologous sequence
  • Genetic recombination method of klebsiella pneumoniae by utilizing short homologous sequence

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preparation example Construction

[0034] Preparation of homologous recombination fragments: two rounds of PCR and one-step cloning.

[0035] The first round of PCR: Design primers according to the description in [Gust B., et al., PCR-targeting system in streptomyces coelicolor A3(2). John Innes Centre: Norwich, 2002.], the 5' end of the primer is short Homologous sequence, use the plasmid carrying the resistance gene as a template to amplify the resistance gene. If the resistance marker needs to be eliminated after recombination, FRT sequences need to be added to both ends of the resistance gene to be amplified.

[0036] Cloning: The fragment amplified by the first round of PCR was connected to the cloning plasmid by TA cloning, transformed into Escherichia coli, and positive recombinants were screened.

[0037] The second round of PCR: design primers upstream and downstream of the cloning site, use the positive recombinants obtained by cloning screening as templates, and amplify to obtain homologous recombina...

example 1

[0043] Using the method of the present invention, the ATP-dependent dihydroxyacetone kinase gene dhak1 is knocked out in the Klebsiella pneumoniae GMCC1.6366 strain (the GMCC1.6366 strain is preserved by the China General Microorganism Collection Center and has ampicillin resistance), Specific steps are as follows:

[0044] 1) Preparation of GMCC1.6366 Competent Cells

[0045] The operation was carried out according to the "production of Klebsiella pneumoniae competent cells" in the specific embodiment described above.

[0046] 2) The pDK6-red plasmid was transferred into GMCC1.6366 strain

[0047] Take 2 μL of the pDK6-red plasmid, mix it with the prepared 100 μL GMCC1.6366 competent cells, set the voltage at 2000 volts, and use a 2 mm electroporation cuvette for electroshock transformation. After transformation, add 1 ml of LB liquid medium to the cells and recover on a 37°C shaker at 200 rpm for 1 hour. Then, spread it on the LB solid plate containing 50mg / L kanamycin, c...

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Abstract

The invention discloses a genetic recombination method of klebsiella pneumoniae by utilizing a short homologous sequence. The method comprises the following steps of: 1) amplifying a resistance gene segment with PCR (polymerase chain reaction), and adding a homologous sequence of a target recombination gene on the primer; 2) transforming the resistance gene segment into colibacillus after the resistance gene segment is connected with a cloned plasmid, and screening to obtain the cloned plasmid; 3) designing a primer by taking the cloned plasmid in the step 2) as a template, and performing PCR amplification to resistance gene, short homologous arms and adjacent upstream and downstream sequences so as to acquire a homologous recombination DNA (deoxyribonucleic acid) segment which is used for knocking out the target gene; 4) transforming the homologous recombination DNA segment to the klebsiella pneumoniae competent cell containing a pDK6-red plasmid; and 5) culturing the transformed klebsiella pneumoniae cell, and screening to obtain a positive recombinant. The method is not required to clone the target recombination gene, is simple and rapid, can be used for carrying out multiple times of gene operation, and has a wide application prospect.

Description

technical field [0001] The invention relates to a gene recombination method, in particular to a method for Klebsiella pneumoniae gene recombination using short homologous sequences. Background technique [0002] Klebsiella pneumoniae (Klebsiellapneumoniae) is a Gram-negative bacterium that is the type species of the genus Klebsiella and belongs to the family Enterobacteriaceae. Klebsiella pneumoniae has the characteristics of fast growth, can use a variety of carbon sources and rich metabolites, and is currently mainly used as bacteria for the production of chemicals such as 2,3-butanediol and 1,3-propanediol. Klebsiella pneumoniae can metabolize 3-hydroxypropionaldehyde, ethanol, acetoin, succinic acid, acetic acid, lactic acid, hydrogen and other chemicals, and Klebsiella pneumoniae can also be used as a host cell to construct 3-hydroxypropionate and other compounds The production of bacteria makes Klebsiella pneumoniae a microorganism with broad industrial application pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N1/20C12R1/22
Inventor 郝健魏东柳鹏福史吉平姜标
Owner SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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