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A suicide plasmid pytklkrt knocking out the prck gene and its construction method

A suicide plasmid and a construction method technology, applied in the field of suicide plasmid and its construction, can solve the problems of cumbersome construction steps, low success rate, short length of homologous fragments, etc., to avoid wrong gene replacement, high success rate, and fewer construction steps. Effect

Active Publication Date: 2016-02-24
HEILONGJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] In order to solve the defects of short length of homologous fragments in the suicide vector, cumbersome construction steps, low success rate and introduction of mutation sites in the construction process of the existing prcK gene knockout vector of Lactobacillus paracasei HD1.7, the present invention provides a A suicide plasmid pYTKLKRT knocking out the prcK gene and its construction method

Method used

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  • A suicide plasmid pytklkrt knocking out the prck gene and its construction method
  • A suicide plasmid pytklkrt knocking out the prck gene and its construction method
  • A suicide plasmid pytklkrt knocking out the prck gene and its construction method

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specific Embodiment approach 1

[0026] Specific embodiment 1: The suicide plasmid pYTKLKRT knocked out the prcK gene of this embodiment is composed of the left fragment prcKL of the prcK gene, the right fragment prcKR of the prcK gene, the tetracycline resistance gene and the plasmid pUC18; the nucleic acid sequence of the fragment prcKL is shown in SEQ ID NO: 1 The nucleic acid sequence of the fragment prcKR is shown in SEQ ID NO: 2.

specific Embodiment approach 2

[0027] Specific embodiment 2: The suicide plasmid pYTKLKRT knocked out prcK gene in this embodiment is constructed according to the following steps:

[0028] 1. Using L.paracaseiHD1.7 genomic DNA as a template, PCR amplifies the left fragment of prcK gene prcKL, the upstream primer of the amplified fragment prcKL is prcKL-up, and the nucleotide sequence of prcKL-up is 5’-CCG GAGCTC TACCTTAATGATTTAGATGCGAGCG-3’, the downstream primer of the amplified fragment prcKL is prcKL-down, and the nucleotide sequence of prcKL-down is 5’-GTC GGTACC GATTGTTCCTTCGGTGTGGATGTGT-3’, the reaction system of PCR amplified fragment prcKL is 50μL from 10μL containing Mg 2+ 5×PrimeSTARBuffer, 4μL of dNTPMixture with a concentration of 2.5mmol / L each, 10μL of L.paracaseiHD1.7 genomic DNA with a concentration of 25ng / μL, 10μL of prcKL-up with a concentration of 1pmol / μL, and 10μL of prcKL with a concentration of 1pmol / μL -down, 0.5μL PrimeSTARHS DNA polymerase 2.5U / μL and 5.5μL sterile ddH 2 O composition...

specific Embodiment approach 3

[0038] Specific embodiment three: this embodiment is different from specific embodiment two in that: in step two, the plasmid pUC18 digestion reaction system is 20μL from 1μL of 10U / μL SacI, 1μL of 10U / μL KpnI, 2μL10× BufferTango, 7μL plasmid pUC18 with a concentration of 80ng / μL and 9μL sterile ddH 2 O composition; digestion at 37°C for 2h, and use the digestion product gel to recover the digested fragments. Other steps and parameters are the same as in the second embodiment.

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Abstract

The invention discloses a prcK gene knocked-out suicide plasmid pYTKLKRT and a construction method thereof, relating to a suicide plasmid and a construction method thereof. The prcK gene knocked-out suicide plasmid pYTKLKRT is composed of a left fragment prcKL of a prcK gene, a right fragment prcKR of the prcK gene, a tetracycline resistance gene and a plasmid pUC18. The construction method comprises the steps: firstly, amplifying the fragment prcKL; secondly, obtaining a plasmid pUC18-KL; thirdly, amplifying the fragment prcKR; fourthly, thus obtaining a plasmid pUC18-KLKR; fifthly, amplifying the tetracycline resistance gene Tet; and sixthly, obtaining the prcK gene knocked-out suicide plasmid pYTKLKRT. The prcK gene knocked-out suicide plasmid pYTKLKRT and the construction method thereof are mainly applied to the field of bioengineering research.

Description

Technical field [0001] The invention relates to a suicide plasmid and a construction method thereof. Background technique [0002] In recent years, researchers have found that in some G + The AIP in the bacterial QS system is not only a signal molecule, it also has antibacterial properties, such as nisin of Lactococcus lactis, phytolactobacter of Lactobacillus plantarum, etc. These antimicrobial peptide (AMP) gene clusters not only contain the ABC transporter coding gene, but also an accessory protein (AP) coding gene, and their products constitute the AMP output and processing system. The structural genes of AMP are located before their corresponding immune protein genes. Most of the AMPs produced are rich in cysteine ​​and are hydrophobic. Moreover, research has found that the maximum production of AMP is related to the non-optimal growth conditions of the producing bacteria. related. [0003] According to the different properties of AMP, AMP can be divided into two categories:...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C12N15/66C12R1/245
Inventor 葛菁萍王洋由田平文祥
Owner HEILONGJIANG UNIV
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