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Streptomyces minitype transposable mutation system as well as construction method and application thereof

A transposase, a pair of technology, applied in the field of biotechnology and genetic engineering, can solve the problems of cumbersome, slow progress of gene function work, low efficiency and so on

Inactive Publication Date: 2011-08-17
FUDAN UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0003] cumbersome and inefficient
Transposon is an effective tool in genetic mutation at present. Researchers have successively discovered and isolated transposons that can be used in Streptomyces. However, some existing transposition systems generally have multiple transpositions, efficiency Low-cost, complicated operation, and heterologous transposons will be modified by the strong restriction modification system of Streptomyces and cannot function, which makes the transposition system in Streptomyces not widely used to screen mutations and elucidate gene functions work is progressing slowly

Method used

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  • Streptomyces minitype transposable mutation system as well as construction method and application thereof
  • Streptomyces minitype transposable mutation system as well as construction method and application thereof
  • Streptomyces minitype transposable mutation system as well as construction method and application thereof

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Embodiment Construction

[0030] Content of the present invention is further described below:

[0031] 1. Obtaining of transposase DNA fragments

[0032] to cultivate Nocardia asteroids spores to extract genomic DNA. Using genomic DNA as a template, PCR amplification was carried out with primers SEQ ID No. 5 and primers SEQ ID No. 6, and the obtained product was subjected to agarose gel electrophoresis, and the resulting band was located below the 1400bp of the control DNA Marker, which was in line with the expected The 1365bp target gene fragment is close in size (including restriction sites and protective bases).

[0033] 2. Construction of pDZY101 Transposition Plasmid

[0034] Saccharopolyspora rubrum Saccharopolyspora erythraea As a template, the two ends of the primers were designed with Xba I and Bam The restriction endonuclease recognition site of HI, the PCR product fragment and the pMD19-T carrier are done T-A clone and then transformed into Escherichia coli, the positive transform...

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Abstract

The invention belongs to the fields of biotechnology and genetic engineering, in particular relates to a high-efficiency streptomyces minitype transposable mutation system as well as a construction method and application thereof. Based on a transposable element sourcing from Norcardia aseteroids YP21, the invention constructs the characteristics of action sites of IS204 transposase in streptomyces coelicolor, and the transposable system is proved to have good genetic stability and good transposable randomness. Transposable mutation is carried out on S. coelicolor M145 by utilizing pDZY101 and derivatives thereof to obtain a great quantity of phenotype mutant strains, in addition, similar characteristics are also found in streptomyces lividans and other streptomyces members, and the characteristics endow the transposable system with good application prospect in aspect of researching type streptomyces functional genes.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and genetic engineering, and specifically relates to a transposition mutation system and its construction method, as well as its application in Streptomyces. Background technique [0002] Use traditional methods such as physical or chemical mutagenesis, site-directed mutagenesis, etc. to discover and study Streptomyces genes [0003] It is cumbersome and inefficient. Transposon is an effective tool in genetic mutation at present. Researchers have successively discovered and isolated transposons that can be used in Streptomyces. However, some existing transposition systems generally have multiple transpositions, efficiency Low-cost, complicated operation, and heterologous transposons will be modified by the strong restriction modification system of Streptomyces and cannot function, which makes the transposition system in Streptomyces not widely used to screen mutations and elucidate gene fun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63C12N15/113
Inventor 丁晓明张欣城鲍芸张渤张霖赵国屏
Owner FUDAN UNIV
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