A kind of pCasPA/pACRISPR two-plasmid system and its application

A plasmid, recombinant system technology, applied in DNA preparation, recombinant DNA technology, introduction of foreign genetic material using vectors, etc., to achieve the effect of broad application prospects

Active Publication Date: 2021-12-31
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, no genome editing has been reported in Pseudomonas aeruginosa

Method used

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  • A kind of pCasPA/pACRISPR two-plasmid system and its application
  • A kind of pCasPA/pACRISPR two-plasmid system and its application
  • A kind of pCasPA/pACRISPR two-plasmid system and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: pCasPA plasmid construction:

[0042] The composition of the pCasPA plasmid is attached figure 1 As shown in A, its sequence is SEQ ID NO:1. The specific construction method of pCasPA plasmid is as follows:

[0043] (1) Using the pKD46 plasmid as a template, the DNA fragment of the λ-Red recombination system and the arabinose-induced P araB The DNA fragment of the promoter; the gene fragment containing the Cas9 protein was amplified by PCR from the pCasSA plasmid.

[0044] The primer sequences used for PCR amplification are:

[0045] Amplified λ-Red recombination system 5' primer sequence (SEQ ID NO: 3):

[0046] 5'-AACGACGGCCAGTGAATTCGAGCTCGGTACCctttcctgcgttGTCGAC

[0047] tgtatttgaaaaataaacaaataggggtcc-3'

[0048] Amplified λ-Red recombination system 3' primer sequence (SEQ ID NO: 4):

[0049] 5'-gtatgaaaagtCTCGAGCATTCTAGAtccacaGCGGCCGCcatggattcttcgtctgtttctactg-3'

[0050] Amplified P araB Promoter 5' primer sequence (SEQ ID NO: 5):

[0051] 5...

Embodiment 2

[0072] Embodiment two: pACRISPR plasmid construction:

[0073] The composition of the pACRISPR plasmid is attached figure 1 As shown in B, its sequence is SEQ ID NO:2. The specific construction method of pACRISPR plasmid is as follows:

[0074] (1) Digest the pAK1900 plasmid with BamHI-HF and HindIII-HF. The reaction system is as follows: 5 μl 10×CutSmart Buffer, 2 μg pAK1900 plasmid, 2 μl BamHI-HF (20 units / μl), 2 μl HindIII-HF (20 units / μl ), and finally add an appropriate amount of ddH 2 O to a total volume of 50 μl. The buffer and enzymes used in this reaction were produced by NEB Company. After the reaction system was reacted at 37° C. for 2 to 3 hours, the double-digested product was purified and recovered using the SanPrep Column PCR Product Purification Kit produced by Sangon Bioengineering (Shanghai) Co., Ltd. The specific steps were carried out according to the operation manual of the kit.

[0075] (2) Using the pEX18Ap plasmid as a template, the DNA fragment o...

Embodiment 3

[0088] Example 3: The pCasPA / pACRISPR dual plasmid system realizes efficient gene knockout in Pseudomonas aeruginosa strains:

[0089] Using the pCasPA / pACRISPR dual plasmid system can achieve efficient knockout of different genes in various strains of Pseudomonas aeruginosa. The process is shown in the attached figure 2 shown. In the experiment, the rhlR gene was selected as an example, and the gene knockout experiment was carried out in Pseudomonas aeruginosa PAO1 strain. attached image 3 A is a schematic diagram of gene knockout in Pseudomonas aeruginosa using pCasPA / pACRISPR dual-plasmid system, and the results of PCR verification, pigment experiment and DNA sequencing of rhlR gene knockout in PAO1 strain.

[0090] (1) First select a DNA fragment of the first 20 bases of a certain NGG (N is any base) sequence on the target gene of the PAO1 strain (these 20 bases are called spacers, and NGG is not included). The feature of this step is: in order to enable the spacer fr...

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Abstract

The present invention provides a pCasPA / pACRISPR dual plasmid system, characterized in that it comprises at least one of pCasPA plasmid and pACRISPR plasmid, the pCasPA plasmid can express Cas9 protein and λ-Red recombination system, and the pACRISPR plasmid Able to express sgRNA. The invention can (1) efficiently and rapidly knock out the genes of various strains of Pseudomonas aeruginosa; (2) efficiently and rapidly perform gene insertion on the genomes of various strains of Pseudomonas aeruginosa. The technology has broad application prospects in the treatment of Pseudomonas aeruginosa infection, drug target discovery, drug development, and physiological research on Pseudomonas aeruginosa.

Description

technical field [0001] The invention relates to a pCasPA / pACRISPR double plasmid system and its application. Background technique [0002] Pseudomonas aeruginosa is a common Gram-negative bacterium that is widely found in the environment, such as soil, water, plants, and the human body, and can cause diseases in patients such as diabetes, severe trauma, cystic fibrosis, cancer, and AIDS. Infection is a common clinical pathogen. Infection by Pseudomonas aeruginosa is difficult to treat, largely due to the synergistic effect of its hypopermeable outer membrane barrier and the resistance-nodulation-cell division (RND) family of multidrug resistance efflux pumps , so that Pseudomonas aeruginosa has strong multi-drug resistance. In recent years, the infection of Pseudomonas aeruginosa has become more and more serious, so it is urgent to find new drug targets and develop new drugs. Genome editing and gene screening technologies are important means to study bacterial pathogenici...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/78C12N1/21C12N15/10C12R1/19C12R1/385
CPCC12N15/102C12N15/78
Inventor 季泉江陈未中
Owner SHANGHAI TECH UNIV
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