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Vibrio mimicus efficient genetic combination method based on natural conversion and application

A technology of mimetic vibrio and genetic recombination, applied in the field of efficient genetic recombination of mimetic vibrio, can solve the problems of low knockout efficiency, low recombination efficiency, cumbersome operation, etc. Effect

Inactive Publication Date: 2017-08-18
SICHUAN AGRI UNIV
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AI Technical Summary

Problems solved by technology

The experimental process of this technology is cumbersome and the recombination efficiency is low
[0003] The λ-RED recombination technology is as follows: 1. Transform the PKD46 plasmid into the target bacterial strain by electroporation, add L-arabinose to culture (PKD46 plasmid can induce the expression of Exo, Beta and Gam proteins through L-arabinose, and Gam proteins can inhibit nuclease degradation Homologous recombination of target DNA mediated by Exo and Beta proteins); 2. Electroporation transformation of target DNA; 3. Screening of positive deletion strains through antibiotic plates; λ-RED recombination technology is based on gene knockout developed by Escherichia coli λ phage Elimination technology is widely used in Escherichia coli or species close to Escherichia coli; Vibrio mimicus will result in low knockout efficiency due to its distant affinity with Escherichia coli; and the operation is cumbersome
[0004] The gene deletion of Vibrio mimicus can now be constructed by λ-RED recombination technology, but its disadvantages are as follows: First, λ-RED recombination technology is based on the gene knockout technology developed by Escherichia coli λ phage, which is widely used in Escherichia coli or Species with close affinity to Escherichia coli; Vibrio mimeticus will result in low knockout efficiency due to its distant affinity with Escherichia coli; Second, the PKD46 plasmid and targeting DNA need to be transformed into the target strain through two electroporations, and the operation process is cumbersome

Method used

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  • Vibrio mimicus efficient genetic combination method based on natural conversion and application
  • Vibrio mimicus efficient genetic combination method based on natural conversion and application
  • Vibrio mimicus efficient genetic combination method based on natural conversion and application

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Step 1. Construction of targeting fragments: Use bacterial genome DNA extraction kit (DP302) to extract the genome of Vibrio mimeticus SCCF01 strain (preserved in the Department of Basic Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, number: CVM2013034), using the genome of SCCF01 strain as a template, Use the target gene upper arm and the target gene lower arm primer pair to amplify the target gene upper arm sequence and lower arm sequence, use the PKD4 plasmid as a template, use the Kan primer pair to amplify the kanamycin resistance gene fragment, and perform overlapping PCR amplification;

[0037] The reaction systems for PCR amplification of the three DNA fragments are: Buffer10μl, dNTPMixture 4μl, upstream and downstream primers (10μM) 1μl each, template 1μl, DNA Polymerase (2.5U / μl) 0.5μl, ddH 2 O32.5ul; PCR amplification conditions: pre-denaturation at 98°C for 3min; denaturation at 98°C for 10s, annealing at 60°C for 10s,...

Embodiment 2

[0052] Embodiment 2 takes TLH as the genetic recombination method of the target gene

[0053] 1. Construction of targeting fragments

[0054] Use bacterial genome DNA extraction kit (DP302) to extract the genome of Vibrio mimicus SCCF01 strain (preserved in the Department of Basic Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, number: CVM2013034), using the SCCF01 strain genome as a template, using TLH-UP-F, R and TLH-DOWN-F, R (Table 1) two pairs of primers respectively amplify TLH upstream and downstream homology arms (nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively); with PKD4 plasmid As a template, Kan-F, R primers (Table 1) were used to amplify the kanamycin resistance gene fragment (nucleotide sequence shown in SEQ ID NO.3). The reaction systems for PCR amplification of the three DNA fragments are: Buffer 10μl, dNTP Mixture 4μl, upstream and downstream primers (10μM) 1μl each, template 1μl, DNA Polyme...

Embodiment 3

[0066] Example 3 Verification of the reliability of Vibrio mimicus genetic recombination method

[0067] In order to further confirm the reliability of the genetic recombination method, this study further constructed the deletion strains of TonB1 single gene, ExbB1-ExbD1 double gene deletion and ExbB1-ExbD1-TonB1 three gene deletion (see Table 2 for the amplification primers); according to Example 1 The method first uses specific primers to amplify the upper arm sequence and lower arm sequence of the target gene, and at the same time amplifies the corresponding kanamycin resistance gene, and then uses the above three fragments as templates to perform overlapping PCR amplification. The obtained PCR product was recovered and purified according to step 3) for natural conversion after gel cutting. Natural Transformation Method:

[0068] a) Inoculate the recipient bacteria on TCBS agar medium, culture at 30°C for 24 hours, pick a single colony and inoculate it in 10ml of LB liquid...

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Abstract

The invention discloses a vibrio mimicus efficient genetic combination method based on natural conversion and application. The method comprises the steps that a vibrio mimicus SCCF01 plant genome is adopted as a template, a target gene upper arm and target gene lower arm primer pair is adopted for amplifying a gene upper arm sequence and a lower arm sequence; a kanamycin resistance gene segment is amplified; the kanamycin resistance gene segment obtained through amplification, the target gene upper arm sequence and the lower arm sequence segments are fused, and a fusion PCR product containing the kanamycin resistance gene segment, the target gene upper arm sequence segments is obtained; the obtained PCR product and the acceptor vibrio mimicus SCCF01 plant are subjected to natural conversion, and the vibrio mimicus SCCF01 plant deleted plant is obtained by screening a resistance marker gene. Compared with the suicide plasmid mediated gene recombination technology and the lambda-RED recombination technology, no electricity conversion plasmid or targeting DNA is needed in the operation process, and operation is easy.

Description

technical field [0001] The invention belongs to the technical field of bacterial genetic transformation, and in particular relates to a natural transformation-based high-efficiency genetic recombination method and application of Vibrio mimeticus. Background technique [0002] The natural transformation (Natural transformation) technology to construct a gene deletion strain was successfully applied to the gene editing of Vibrio cholerae in 2010, and has been continuously improved since then. The theoretical basis of this method is based on the principle of natural transformation of bacteria. Under the condition of nutrient starvation, Vibrio can become naturally competent to absorb foreign DNA through chitin induction. When the foreign DNA is homologous to its own target gene, it can Homologous recombination exchange occurs with bacterial target genes. However, this technique has not been successfully applied to Vibrio mimicus. The method of gene deletion also includes: gen...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N1/21C12R1/63
CPCC12N15/90
Inventor 耿毅余泽辉邓钊宾汪开毓陈德芳欧阳萍黄小丽杨泽晓
Owner SICHUAN AGRI UNIV
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