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prcK gene knocked-out suicide plasmid pYTKLKRT and construction method thereof

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

Active Publication Date: 2014-03-05
HEILONGJIANG UNIV
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  • Abstract
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  • Application Information

AI Technical Summary

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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  • prcK gene knocked-out suicide plasmid pYTKLKRT and construction method thereof
  • prcK gene knocked-out suicide plasmid pYTKLKRT and construction method thereof
  • prcK gene knocked-out suicide plasmid pYTKLKRT and construction method thereof

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specific Embodiment approach 1

[0026] Embodiment 1: The suicide plasmid pYTKLKRT for knocking out the prcK gene in this embodiment is composed of prcKL, a fragment on the left side of the prcK gene, prcKR, a fragment on the right side of the prcK gene, a tetracycline resistance gene, and plasmid pUC18; the nucleic acid sequence of the fragment prcKL is as 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 two: The suicide plasmid pYTKLKRT knocking out the prcK gene in this embodiment is constructed according to the following steps:

[0028] 1. Use L.paracasei HD1.7 genomic DNA as a template to PCR amplify the left fragment prcKL of the prcK gene, 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 the PCR amplified fragment prcKL is 50 μL by 10 μL containing Mg 2+ 5×PrimeSTAR Buffer, 4μL of dNTP Mixture with a concentration of 2.5mmol / L, 10μL of L.paracasei HD1.7 genomic DNA with a concentration of 25ng / μL, 10μL of prcKL-up with a concentration of 1pmol / μL, 10μL of a concentration of 1pmol / μL µL of prcKL-down, 0.5 µL of PrimeSTAR HS DNA polymerase at a concentration of 2.5 ...

specific Embodiment approach 3

[0038] Specific embodiment three: the difference between this embodiment and specific embodiment two is: in step two, the enzyme digestion reaction system of plasmid pUC18 is 20 μL consisting of 1 μL of Sac I with a concentration of 10 U / μL, 1 μL of Kpn I with a concentration of 10 U / μL, 2 μL 10×Buffer Tango, 7 μL plasmid pUC18 with a concentration of 80 ng / μL and 9 μL sterile ddH 2 O composition; digest at 37°C for 2 h at constant temperature, and use enzyme 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 its construction method. Background technique [0002] In recent years, researchers have found that in certain G + AIP in the bacterial QS system is not only a signal molecule, but also has antibacterial ability, such as nisin of Lactococcus lactis, phytolactin of Lactobacillus plantarum and so on. These antimicrobial peptide (AMP) gene clusters contain not only ABC transporter encoding genes, but also an accessory protein (AP) encoding gene, and their products constitute the AMP export and processing system. The structural genes of AMP are all located in front of their corresponding immune protein genes. Most of the AMP produced is rich in cysteine ​​and has hydrophobicity. Moreover, it has been found through research 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 AMPs, AMPs can be ...

Claims

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Application Information

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Patent Type & Authority Applications(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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