Preparation method of Nocardia competent cells and gene editing method of Nocardia

A technology of competent cells and Nocardia, applied in the field of molecular biology, to achieve high application value, simple and fast operation

Active Publication Date: 2019-08-16
SHENZHEN INST OF GUANGDONG OCEAN UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no relevant research to apply CRISPR / Cas9 technology to Nocardia

Method used

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  • Preparation method of Nocardia competent cells and gene editing method of Nocardia
  • Preparation method of Nocardia competent cells and gene editing method of Nocardia

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preparation example Construction

[0036] The present invention firstly provides a kind of preparation method of Nocardia competent cell, comprising:

[0037] Take the Nocardia bacterial liquid in the logarithmic growth phase in a centrifuge tube, collect the Nocardia bacterial cells by centrifugation for the first time, wash the bacterial cells with sterile water or sterile aqueous solution, and then use sterile Water or a sterile aqueous solution is used to resuspend the bacteria for the first time, and then add lysozyme to the resuspended bacteria for treatment. After the treatment, the bacteria of Nocardia are collected by centrifugation for the second time. The bacteria aqueous solution was used to resuspend the bacteria for the second time to obtain Nocardia competent cells.

[0038] Preferably, after the Nocardia bacteria are collected by centrifugation for the first time, the bacteria are washed with sterile water for several times, and then the bacteria are resuspended with sterile water for the first ...

Embodiment 1

[0078] pCRISPomyces-2 plasmid transformation

[0079] Step 1. With the help of the CRISPR online design tool (http: / / www.e-crisp.org / E-CRISP / ), find out the appropriate action site PAM position of the 2816 and 3141 genes in the genome of the yellowtail Nocardia According to the PAM site, an appropriate sgRNA anchor sequence is designed, and the sgRNA anchor sequence is a 20bp sequence upstream of the PAM site. The sgRNA anchor sequence of the 2816 gene designed according to this step can be any one of SEQ ID NO.2-8, and the one used in the embodiment of the present invention is SEQID NO.2; the sgRNA anchor sequence of the 3141 gene can be Any one of SEQ ID NO.10-17, used in the embodiment of the present invention is SEQ ID NO.10.

[0080] Step 2: Synthesize a 24bp oligonucleotide fragment according to the designed sgRNA anchor sequence, and the primers for the synthetic oligonucleotide fragment (synthesized by Guangzhou Aiji Biological Co., Ltd.) include upstream ACGC cohesiv...

Embodiment 2

[0090] Preparation of Competent Cells of Nocardia from Yellowtail

[0091] Step 1. Aseptically inoculate the preserved Nocardia spp. into BHI solid culture medium (purchased from Guangdong Huankai Microbiology Technology Co., Ltd.), and incubate it upside down at 28°C. After a single colony grows on the plate, pick a single colony into 50ml of BHI liquid medium by aseptic operation, and cultivate it at 28°C and 130rpm until the logarithmic growth phase.

[0092] Step 2. Take 30ml of Nocardia japonica that has reached the logarithmic growth phase in a 50ml centrifuge tube, collect the bacterial cells at 4°C and 8000rpm; wash the bacterial cells twice with 10ml of sterile water, and then use 10ml of sterile water Resuspend the bacterial cells, then add an appropriate amount of lysozyme (purchased from Guangzhou Dongsheng Biotechnology Co., Ltd.) to the centrifuge tube and mix evenly so that the final mass concentration of lysozyme is 5 mg / ml; Gently shake the centrifuge tube ev...

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Abstract

The invention provides a preparation method of Nocardia competent cells and a gene editing method of Nocardia. According to the preparation method, Nocardia is treated with lysozyme so that the thickness of cellular wall of Nocardia is deceased; Nocardia competent cells easily accepting exogenous DNAs are finally prepared. The preparation method is simple and quick to perform and has high application value. A CRSIPR / Cas9 system is applied to Nocardia for the first time herein according to the gene editing method of Nocardia; a CRISPR / Cas9 genetic editing plasmid is constructed and is convertedand delivered to Nocardia for editing a target gene. The gene editing method is simple and quick to perform, has high application value and helps lay the basis for the study on gene functionality ofNocardia, the construction of gene deleted strains of Nocardia and the preparation of gene deleted vaccines of Nocardia.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a preparation method of Nocardia competent cells and a Nocardia gene editing method. Background technique [0002] Nocardiosis in yellowtail is the main pathogenic bacterium of nocardiosis in aquaculture. When fish have low immunity, they can be infected through bait, gills or wounds. Taxonomically, Nocardia spp. belongs to Actinomycetes, Nocardiaceae, and Nocardia genus, and is a Gram-positive bacterium. Nocardiella yellowtail is a facultative intracellular parasite. During its infection, the bacteria will be phagocytized by fish phagocytic cells, mainly macrophages. Nocardia spp. can survive in macrophages, and can spread and infect various organs of fish with the migration of macrophages. The bacteria can mainly cause white nodules in the internal organs of fish, resulting in slow movement of fish, loss of appetite, low immunity, and death. Yellowtail Nocardia can infect mo...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/21C12N15/74C12R1/365
CPCC12N1/20C12N15/74C07K14/355Y02A50/30
Inventor 夏立群王文基鲁义善侯素莹谭万春陈建林
Owner SHENZHEN INST OF GUANGDONG OCEAN UNIV
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