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Suicide plasmid pYTRLRRT with prcR gene knockout effect and construction method thereof

A technique of suicide plasmid and construction method, which is applied in the field of suicide plasmid and its construction, can solve the problems such as failure to successfully knock out the PRCR gene of Lactobacillus paracasei, achieve the goal of increasing homologous recombination rate, avoiding wrong gene replacement, and easy screening Effect

Active Publication Date: 2014-02-19
HEILONGJIANG UNIV
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  • Application Information

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Problems solved by technology

[0008] In order to solve the problem that the existing suicide plasmids fail to successfully knock out the prcR gene in Lactobacillus paracasei (Lactobacillus paracasei) HD1.7 cells, the present invention provides a suicide plasmid pYTRLRRT for knocking out the prcR gene and its construction method

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  • Suicide plasmid pYTRLRRT with prcR gene knockout effect and construction method thereof
  • Suicide plasmid pYTRLRRT with prcR gene knockout effect and construction method thereof
  • Suicide plasmid pYTRLRRT with prcR gene knockout effect and construction method thereof

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specific Embodiment approach 1

[0025] Embodiment 1: The suicide plasmid pYTRLRRT for knocking out the prcR gene in this embodiment is composed of prcRL, the left fragment of the prcR gene, prcRR, the right fragment of the prcR gene, a tetracycline resistance gene, and plasmid pUC18; the nucleic acid sequence of the prcRL fragment is as shown in SEQ ID NO: 1; the nucleic acid sequence of the fragment prcRR is shown in SEQ ID NO:2.

specific Embodiment approach 2

[0026] Specific embodiment two: The suicide plasmid pYTRLRRT knocking out the prcR gene in this embodiment is constructed according to the following steps:

[0027] 1. Use L.paracasei HD1.7 genomic DNA as a template to PCR amplify the left fragment prcRL of the prcR gene, the upstream primer of the amplified fragment prcRL is prcRL-up, and the nucleotide sequence of prcRL-up is 5'-CCG GAGCTC CCTACTCAGCATTCAGAGGTCAACT-3', the downstream primer of the amplified fragment prcRL is prcRL-down, and the nucleotide sequence of prcRL-down is 5'-GTC GGTACC ATCTTCAGCATCGTTTGGTGGTTGG-3', the reaction system of the PCR amplified fragment prcRL 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 prcRL-up with a concentration of 1pmol / μL, and 10μL of a concentration of 1pmol / μL µL of PRCRL-down, 0.5 µL of PrimeSTAR HS DNA polymerase at a concentration of ...

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 provides a suicide plasmid pYTRLRRT with a prcR gene knockout effect and a construction method thereof, and relates to a suicide plasmid and a construction method thereof. The suicide plasmid pYTRLRRT with the prcR gene knockout effect is formed by a prcR gene left fragment prcRL, a prcR gene right fragment prcRR, a tetracycline resistant gene and a plasmid pUC18. The construction method comprises the steps as follows: 1, amplifying the fragment prcRL; 2, obtaining a plasmid pUC18-RL; 3, amplifying the fragment prcRR; 4, obtaining a plasmid pUC18-RR; 5, amplifying the tetracycline resistant gene Tet; and 6, obtaining the suicide plasmid pYTRLRRT with the prcR gene knockout effect. The invention is applied to the field of the 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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IPC IPC(8): C12N15/63C12N15/66
Inventor 葛菁萍王洋由田平文祥
Owner HEILONGJIANG UNIV
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