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A group of Lactobacillus casei gene knockout vectors and applications thereof

A technology of Lactobacillus casei and gene knockout, which is applied in the field of molecular biotechnology to achieve the effect of improving efficiency and shortening the cycle

Active Publication Date: 2020-07-31
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, after searching, the special vector pMSP456 that mediates the homologous recombination between the exogenous double-stranded DNA knockout cassette (up-lox66-cat-lox71-down) and the homologous sequence on the chromosome and mediates the lox66 and lox71 sites The recombination reaction between the special carrier pMSPcre and its application patent for excision of the resistance gene has not been reported

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  • A group of Lactobacillus casei gene knockout vectors and applications thereof
  • A group of Lactobacillus casei gene knockout vectors and applications thereof
  • A group of Lactobacillus casei gene knockout vectors and applications thereof

Examples

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Embodiment 1

[0067] Example 1: Construction of two novel Lactobacillus casei gene knockout-specific vectors

[0068] (1) Amplification primer design (Beijing Dingguochangsheng Biotechnology Co., Ltd., PAGE purification).

[0069] Primers used to amplify LCABL_13040-50-60:

[0070] 456F: 5'-AA CTGCAG ATGACCATGCTTGATTACAACACAG-3'

[0071] 456R: 5'-AA TCTAGA CTACTCGACTAGCTCATCCATGCT-3'

[0072] Primers used to amplify cre:

[0073] creF: 5'-AA CTGCAG ACATGTCCAATTTACTGACCGTAC-3'

[0074] creR: 5'-ACGC GTC GAC TTAATCGCCATCTTCCAGCA-3'

[0075] The underlined sequence represents the added restriction site:

[0076] PstI: CTGCAG;

[0077] XbaI: TCTAGA;

[0078] SalI:GTCGAC.

[0079] After synthesis, use ultrapure water to prepare at a concentration of 10 μM at -20°C for later use.

[0080] (2) LCABL_13040-50-60 and cre fragments were obtained.

[0081] Amplify using 2×Primestar Max (TaKaRa) in sterile 0.2mL PCR reaction tubes, the system is as follows:

[0082]

[0083] After m...

Embodiment 2

[0111] Example 2: Application of Lactobacillus casei Gene Knockout Vector in Construction of Lactobacillus Casei Gene Knockout Mutants of the Present Invention and Identification of Knockout Efficiency

[0112] (1) Transfer the Lactobacillus casei BL23 activated by MRS overnight into 5mL SMRS at a 2% inoculum size, culture statically at 37°C until OD600=0.30-0.60, make competent cells and transform the vector pMSP456 by electroporation, and after 2 hours of recovery, apply Spread on the MRS plate with a final concentration of 5 μg / mL erythromycin. Pick erythromycin-resistant transformants, extract plasmids, and use pMSP456 specific primers (pMSPF and pMSPR) to detect whether the vector has been transferred by PCR. The results are as follows: figure 1 .

[0113](2) After the correct transformants verified in step (1) were activated overnight, they were transferred to SMRS (with a final concentration of 5 μg / mL erythromycin) according to the inoculum size of 4%, and cultured s...

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Abstract

The invention discloses a Lactobacillus casei gene knockout vector group. The Lactobacillus casei gene knockout vector group comprises a special vector pMSP456 which mediates the homologous recombination between a foreign double-stranded DNA knockout box up-lox66-cat-lox71-down and a homologous sequence on a chromosome, and a special vector pMSPcre which mediates the recombination reaction between loci lox66 and lox71 in order to excise a resistant gene. The nucleotide sequence of the vector pMSP456 is shown in SEQ ID No. 1, and the nucleotide sequence of the vector pMSPcre is shown in SEQ ID No. 2. The invention further discloses the application of the vectors to construction of a Lactobacillus casei gene knockout mutant strain. Experiments show that by using the vectors pMSP456 and pMSPcre to knock out a Lactobacillus casei gene, knockout efficiency reaches 100%, operation is easy, time consumption is low, and the vectors have broad application prospects in Lactobacillus casei metabolic engineering.

Description

technical field [0001] The invention belongs to the field of molecular biology technology, relates to a set of special vectors for gene knockout, in particular to mediating homologous sequences between exogenous double-stranded DNA knockout cassettes (up-lox66-cat-lox71-down) and homologous sequences on chromosomes. A special vector for source recombination and a special vector for mediating the recombination reaction between lox66 and lox71 sites so as to excise the resistance gene, and its application in the efficient and rapid construction of Lactobacillus casei gene knockout mutants. Background technique [0002] Lactobacillus casei is a Gram-positive bacterium widely used in dairy processing. At present, Lactobacillus casei has been found to have significant probiotic effects on the host, such as regulating inflammation, promoting immunity, improving gastrointestinal dysfunction, preventing allergies, etc., so it is often used as a probiotic in the development and produ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C12N1/21C12R1/245
CPCC07K14/335C12N15/74
Inventor 孔健郭婷婷辛永平
Owner SHANDONG UNIV