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Strong promoter-containing plasmid for traceless editing of streptomyces gene

A strong promoter and gene editing technology, which is applied in genetic engineering, plant genetic improvement, and the use of vectors to introduce foreign genetic material, etc., can solve the problem of inability to realize gene editing without trace operation

Inactive Publication Date: 2022-03-22
ZUNYI NO 1 PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional Streptomyces gene knockout method is limited by resistance selection markers and cannot realize the traceless operation of gene editing, and the residue of the marker gene will bring uncontrollable impact on further research and application

Method used

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  • Strong promoter-containing plasmid for traceless editing of streptomyces gene
  • Strong promoter-containing plasmid for traceless editing of streptomyces gene
  • Strong promoter-containing plasmid for traceless editing of streptomyces gene

Examples

Experimental program
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Effect test

Embodiment

[0038] Such as figure 1 As shown, the TCRAS system was constructed based on the following principles, and the expression regulation mode of the toxin-antitoxin mazEF system controlled by the promoter was changed: the expression of the antitoxin MazE was induced by an inducible promoter, the toxin MazF was continuously expressed by a strong constitutive promoter, and the antitoxin switch regulated Toxin counter-selection-cassette regulated by an antitoxin switch (TCRAS). Adding an inducer under normal conditions, the antitoxin toxin MazE can neutralize the toxicity of the toxin MazF; under reverse screening conditions, without an inducer, the free MazF cleaves single-stranded RNA at the ACA site of the RNA, organizes protein translation, and results in a TARAS-containing system The cells died, while the cells deleted by homologous recombination of TARAS were not affected, and grew normally, achieving reverse screening.

[0039] Such as figure 2 and image 3 As shown, the St...

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Abstract

The invention relates to a strong promoter-containing plasmid for streptavidin gene traceless editing, which comprises: a strong promoter Psco5768 and a codon-optimized toxin gene mazF, the strong promoter Psco5768 controls the expression of the codon-optimized toxin gene mazF, the strong promoter Psco5768 controls the expression of the codon-optimized toxin gene mazF, and the strong promoter Psco5768 controls the expression of the codon-optimized toxin gene mazF; the sequence of the strong promoter Psco5768 comprises a sequence as shown in SEQ ID NO: 4, and the sequence of the codon-optimized toxin gene mazF comprises a sequence as shown in SEQ ID NO: 3. The plasmid constructed by the invention selects a promoter with proper strength, and can be used for efficient traceless gene editing plasmids for multiple edits such as streptomycete gene knockout, large fragment knockout, site-specific mutagenesis, gene insertion, large fragment insertion and the like, and an efficient tool is provided for development of natural products from streptomycete.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a streptomyces gene scarless editing plasmid containing a strong promoter. Background technique [0002] Promoter is a DNA sequence that RNA polymerase recognizes, binds and initiates transcription, and contains conserved sequences required for specific binding of RNA polymerase and initiation of transcription. Promoters can affect gene expression by increasing or decreasing the rate of gene transcription. Therefore, the selection of a suitable promoter is critical for gene expression. [0003] Streptomyces are a major source of bioactive natural products, but relatively inefficient genetic manipulation methods have greatly hindered the metabolic engineering of natural products from these organisms. The traditional Streptomyces gene knockout method is limited by resistance selection markers and cannot realize the traceless operation of gene editing, and the residue of m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/76C12N15/31C12N1/21C12R1/465
CPCC12N15/76C07K14/195C12N2830/55C12N2830/34C12N2800/22
Inventor 吴杰曲璟秋叶萌邓成敏李晓倩
Owner ZUNYI NO 1 PEOPLES HOSPITAL