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DNA (deoxyribonucleic acid) molecule for constructing coxiella burnetii inducible CRISPRi (clustered regularly interspaced short palindromic repeats i) system and application

A technology of C. bainische and DNA molecules, applied in the field of genetic engineering, can solve the problems of cumbersome operation, low incidence of homologous recombination, and cost.

Active Publication Date: 2022-07-22
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, this technology is cumbersome to operate, and it needs to use antibiotics and sucrose as selection markers to screen positive clones twice; on the other hand, due to the low incidence of homologous recombination of Coxiella beinii, recombination plasmids with different lengths of homologous arms need to be designed Try it out, usually a successful gene knockout experiment takes several months

Method used

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  • DNA (deoxyribonucleic acid) molecule for constructing coxiella burnetii inducible CRISPRi (clustered regularly interspaced short palindromic repeats i) system and application
  • DNA (deoxyribonucleic acid) molecule for constructing coxiella burnetii inducible CRISPRi (clustered regularly interspaced short palindromic repeats i) system and application
  • DNA (deoxyribonucleic acid) molecule for constructing coxiella burnetii inducible CRISPRi (clustered regularly interspaced short palindromic repeats i) system and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1. Construction of an inducible CRISPRi system

[0068] 1.1. Construction of pdCas9 plasmid

[0069] (1) dCas9 gene synthesis

[0070] GenScript Biotechnology Co., Ltd. was entrusted to artificially synthesize the dCas9 gene of Streptococcus pyogenes. The nucleotide sequence of the dCas9 gene is SEQ ID No.1, of which the 1-6th position of SEQ ID No.1 is the BglII enzyme cleavage site, Positions 7-18 are RBS, positions 25-4131 are dCas9 gene, and positions 4132-4137 are SalI restriction sites.

[0071] (2) teto-TetR sequence synthesis

[0072] Entrust GenScript Biotechnology Co., Ltd. to synthesize the tetO-TetR sequence. The nucleotide sequence of the synthesized sequence is SEQ ID No.2, of which the 1-6th position of SEQ ID No.2 is the EcoRI restriction site , the 7-630th position is the reverse complement of Tet repressor protein (TetR), the 649th-704th position is the Tet operon (Tetoperator, TetO), and the 716th-721st position is the BglII restriction sit...

Embodiment 2

[0092] Example 2. Detection of the efficiency of inducible CRISPRi system in inhibiting the expression of dotB in Kirkkiella

[0093] 2.1. Preparation of DotB and Com1 protein antiserum

[0094] (1) Preparation of Cokes body dot B gene and com 1 gene prokaryotic expression vector, dot The nucleotide sequence of the coding sequence of the B gene is SEQ ID No. 4, com The nucleotide sequence of the 1 gene is SEQ ID No.5.

[0095] (2) Insert the above sequences into the multiple cloning sites of the prokaryotic expression vector pET32a(+) respectively, and transform into E. coli BL21 (DE3) competent cells (Beijing Quanshijin Biotechnology Co., Ltd., product number CD601-02) , and then cultured in LB liquid medium at 37 °C and 200 r / min for more than 14 h. Then transfer into LB liquid medium containing ampicillin resistance at a volume ratio of 1:100, and cultivate to OD at 37 °C and 200 r / min. 600 = 0.6, then IPTG was added to make the final concentration in the system 5...

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Abstract

The invention discloses a DNA molecule for constructing a coxiella burnetii inducible CRISPRi (clustered regularly interspaced short palindromic repeats i) system and application of the DNA molecule, and belongs to the technical field of genetic engineering. The invention provides a DNA molecule. The DNA molecule sequentially comprises a promoter, an sgRNA gene, an antisense gene of Tet repressor protein, a Tet operon, a dCas9 gene and a reverse promoter of the promoter from upstream to downstream, and the reverse promoter is a promoter of which the nucleotide sequence is reversely complementary with the nucleotide sequence of the promoter. According to the method, expression inhibition of the specific gene of the coxiella burnetii can be completed only by transferring plasmids containing the DNA molecules into the coxiella burnetii at a time, and the method can be used for performing genetic manipulation on the coxiella burnetii more conveniently and quickly; and a technical basis is provided for researching biological functions and toxicity phenotypes of specific genes of coxiella burnetii.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a DNA molecule and an application for constructing an inducible CRISPRi system of C. beriberi. Background technique [0002] C. baishii ( Coxiella burnetii ) is the pathogen of an important zoonotic disease Q fever, which is mainly excreted from the body through the abortion or childbirth excrement, feces, vaginal secretions, milk, etc. of infected animals, forming aerosols containing Coxoidella, causing human infection. [0003] DotB is a chaperone protein of the type IV secretion system of C. beijerii. Knocking out or knocking down the expression of this protein does not affect the growth and reproduction level of C. beijerii in the medium in vitro, but it will affect the growth and reproduction level of C. beijerii. The growth and reproduction level of the body in the host cell; Com1 is a 27kDa outer membrane protein of C. beijerii, an essential protei...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/85C12N15/113C12N15/55C12N15/66C12N1/21C12N5/10C12R1/01
CPCC12N15/74C12N15/85C12N15/113C12N15/66C12N9/22C12N2310/20C12N2830/006
Inventor 熊小路焦俊张家宁付梦姣欧阳譞于永慧赵明亮张珊周春雨
Owner ACADEMY OF MILITARY MEDICAL SCI
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