Construction and usage of lactobacillus single-plasmid Nisin inducible expression vector

A technology of inducing expression and lactic acid bacteria, applied in the biological field, can solve the problems of restriction, low efficiency of host chromosome integration, narrow vector host spectrum, etc.

Inactive Publication Date: 2010-02-03
王春凤 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the main drawback is that the host chromosome integration efficiency of the nisRK gene is low, and the same treatment needs to be carried out for each host strain selected. The genetic manipulation is cumbersome, and the vector host spectrum is narrow, which restricts its application.

Method used

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  • Construction and usage of lactobacillus single-plasmid Nisin inducible expression vector
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  • Construction and usage of lactobacillus single-plasmid Nisin inducible expression vector

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Experimental program
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Effect test

Embodiment 1

[0108] (1) Acquisition and identification of the two-component regulatory element nisRK gene

[0109] 1 Main test materials and molecular biology reagents

[0110] (1) Strains and plasmid vectors

[0111] Lactococcus lactis producing Nisin, E.coli JM109 competent cells; pGEM-T plasmid vector.

[0112] (2) Tool enzymes and main reagents

[0113] Pfu DNA Polymerase, T4 DNA Ligase, Xba I and Kpn I Restriction Enzyme, Ex Taq DNA Polymerase, dNTP, RNase, DNA A-Tailing Kit, DNA Gel Extraction Kit, Chromosome Extraction Kit , plasmid extraction reagents, ampicillin, etc.

[0114] (3) culture medium

[0115] MRS medium for lactic acid bacteria, LB Escherichia coli medium, LB medium containing ampicillin.

[0116] 2 Preparation of Nisin-producing Lactococcus lactis chromosomal DNA

[0117] Chromosomal DNA of Lactococcus lactis was extracted using a chromosome extraction kit according to the instructions, and 2 μL was used for detection by 0.8% agarose gel electrophoresis. As a r...

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Abstract

The invention provides construction and usage of lactobacillus single-plasmid Nisin inducible expression vector, which belongs to the field of biotechnology. The construction comprises: a, acquisition and identification, the preparation of chromosome DNA producing Nisin lactococcus lactis, the design and synthesis of primer, the PCR amplification, recovery, pGEM-T connection and transformation ofa nisRK gene, and the identification of the nisRK gene; and b, the construction of pW425N, the recovery, connection and identification of the nisRK gene and a basic vector pW425et, the acquisition and identification of inducible promoter nisA gene, and the construction of pW425N. The usage comprises: a, the acquisition and identification of a green fluorescent protein gfp gene; b, the construction and identification of a recombinant expression vector; c, the determination of the inducible expression function of a Nisin inducible expression vector for green fluorescent protein; and d, the detection of lactobacillus stability. The invention has the advantages of avoiding the troublesome operation of integrating chromosomes of host bacteria in the prior system and simplifying the construction of recombinant genetic-engineering lactic acid bacteria.

Description

Technical field: [0001] The invention belongs to the field of biotechnology. Background technique [0002] 1 Definition and distribution of lactic acid bacteria [0003] Lactic acid bacteria (LAB) is a general term for a group of bacteria that can ferment carbohydrates to produce large amounts of lactic acid. This type of bacteria is Gram-positive, spherical or rod-shaped, catalase-negative, consumes more than 50% of glucose to produce lactic acid; does not form spores, does not move or has weak motility; can decompose proteins, but not Produce spoilage products; weak ability to decompose fat. [0004] Lactic acid bacteria are widely distributed in nature, including the digestive system, respiratory system, genitourinary system, oral system, skin system and excrement of humans and animals, milk and dairy products, fruits, roots and branches of plants, rotten plants, Fermented animal foods and beverages, compost, soil and sludge, sewage, several clinical samples, etc. [2]...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12Q1/02G01N21/64
Inventor 王春凤郝凤奇
Owner 王春凤
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