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Plasmid for CRISPRi system, construction method of plasmid, and application of plasmid to directional silencing of yersinia pestis target gene

A construction method and gene technology, applied in gene editing and biological fields, can solve problems such as complex operation and low efficiency, and achieve the effect of reducing transcription level and protein level

Active Publication Date: 2019-06-07
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the research on Yersinia pestis, the traditional gene knockout technology is based on the principle of homologous recombination. In recent years, reports have confirmed the feasibility of the application of the CRISPR / Cas system in Yersinia pestis ( Yan, M.Y., Yan, H.Q., Ren, G.X., Zhao, J.P., Guo, X.P., & Sun, Y.C. (2017). CRISPR-Cas12a-Assisted Recombineering in Bacteria. Appl Environ Microbiol, 83(17).), these gene editing techniques It can realize gene knockout in Yersinia pestis and modify and edit its genome. Although it can be used to study the gene function of Yersinia pestis, it has the disadvantages of complex operation, low efficiency, and irreversibility

Method used

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  • Plasmid for CRISPRi system, construction method of plasmid, and application of plasmid to directional silencing of yersinia pestis target gene
  • Plasmid for CRISPRi system, construction method of plasmid, and application of plasmid to directional silencing of yersinia pestis target gene
  • Plasmid for CRISPRi system, construction method of plasmid, and application of plasmid to directional silencing of yersinia pestis target gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: sgRNA expression plasmid and pdCas9-tetO plasmid

[0045] 1.1. Construction of the plasmid pgRNA-phoP: Use the pgRNA vector (empty vector, its schematic structure diagram is shown in 1A, obtained from Addgene#44251, website: https: / / www.addgene.org / ) as a template, using Table 1 below The primers phop-1o-F (SEQ ID NO: 21) and phop-1o-R (SEQ ID NO: 22) shown in the pgRNA vector were compared to the pgRNA vector according to the system shown in Table 2 below and the PCR program shown in Table 3 below. Perform PCR amplification to obtain PCR amplified fragments, and then use restriction nucleases Spe I and HindIII to digest the pgRNA vector and its PCR amplified fragments according to the enzyme digestion system shown in Table 4 below, and then use T4 ligase The ligation system shown in Table 5 below connects the two digested fragments to obtain the sgRNA expression plasmid (control plasmid) for the target gene phoP of Yersinia pestis, which is named plasmid pgRNA...

Embodiment 2

[0086] Example 2: Use of inducer

[0087] 2.1. The inventors selected 9 genes (ail, cobB, cspB, hdeD, hmsH, ibpB, pla, slyA, yscB) located on the chromosome or plasmid in Yersinia pestis phenotype. The function of the gene is shown in Table 10 above), and the sgRNA expression plasmid (see Table 10) constructed in Example 1 and the dCas9-tetO plasmid are transformed into bacteria by electric shock to construct the corresponding target gene silenced strain, and use 1μg / ml of aTc is an inducer. The constructed silent strains are divided into silent strains under induction conditions (the inducer is added at the time of transformation) and silent strains under non-induction conditions. At the same time, a wild control strain is established. Each strain is cultivated to mid-log phase, namely OD 620 Is around 1.0.

[0088] Use the nucleotide sequence shown in Table 12 above to test the target gene transcription level (expressed in mRNA) for each strain tested in qRT-PCR (real-time fluo...

Embodiment 3

[0092] Example 3: Protein level expression

[0093] The present inventors selected 4 target genes hmsH, phoP, pla, and slyA of Yersinia pestis (see Table 10 above for the function of each gene), and used the double plasmid system corresponding to the target genes constructed in Example 1 to transform into the plague by electric shock To construct the corresponding target gene-silencing strain, and to induce culture under non-inducing conditions and 1μg / ml aTc as the inducing agent (the inducer is added at the time of transformation), and the wild strain of Yersinia pestis is established at the same time As a control, each strain of the test was cultured to mid-log phase, namely OD 620 Is around 1.0. Western blotting was used to verify the protein level changes of the target gene between the wild strain and the corresponding silent strain, and the GroEL protein was used as an internal reference to maintain the same amount of sample. The result is Figure 5 As shown in the figure...

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Abstract

The invention disclose a plasmid for CRISPRi system, a construction method of the plasmid, and an application of the plasmid to directional silencing of yersinia pestis target gene, and belongs to thetechnical field of gene editing. The plasmid contains a sgRNA expression plasmid and a pdCas9-tetO plasmid. A constructed dual-plasmid CRISPRi system is transformed into yersinia pestis, and in the presence of a revulsant namely anhydrotetracycline hydrochloride, the target gene can be in directional silencing. The CRISPRi system can substantially reduce the transcriptional level and the proteinlevel of the target gene, can enable the yersinia pestis to show the relevant phenotype of the deficiency target gene, and can be used as a novel tool for high-flux gene function research in the yersinia pestis.

Description

Technical field [0001] The invention belongs to the field of biotechnology, in particular to the technical field of gene editing, and specifically relates to a plasmid used in the CRISPRi system, a construction method thereof, and its application in targeted silencing of Y. pestis target genes. Background technique [0002] Yersinia pestis (Yersinia pestis, hereinafter also referred to as Yersinia pestis) is the pathogen of plague, a natural foci of a powerful infectious disease. There was a record of the plague epidemic as early as 2000, which caused three pandemics worldwide and brought huge disasters to mankind. [0003] The plague is a statutory Class A infectious disease in my country, and the Yersinia pestis is also one of the Class A biological warfare agents with strong infectiousness and high pathogenicity. With the improvement of the level of epidemic prevention, the incidence of plague has been controlled, but in terms of treatment, there is no other effective treatment...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/66C12N9/22
Inventor 王桐杜宗敏杨瑞馥宋亚军
Owner ACADEMY OF MILITARY MEDICAL SCI
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