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High-efficiency transposable mutation system and construction method

A technology of transposase and transposon, applied in the field of molecular biology, can solve the problems of cumbersome operation, slow progress in screening mutant strains and research on corresponding gene functions, and lack of general application, etc.

Active Publication Date: 2018-11-02
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing transposon systems used in yeast generally have problems such as low transposition efficiency, low randomness of insertion mutations, and cumbersome operations, which make the transposon mutation system in yeast not widely used. Slow progress in screening mutants and investigating corresponding gene functions

Method used

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  • High-efficiency transposable mutation system and construction method
  • High-efficiency transposable mutation system and construction method
  • High-efficiency transposable mutation system and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Embodiment 1, construction of auxiliary plasmid pBRZeo-TTcBaseCOV596A and pBRZeo-MSBase100X

[0104] Extract Pichia total genomic DNA, extract pGALS-TcBuster CO V596A (from Johns Hopkins University School of Medicine), pCMV(CAT)T7-SB100 (from Addgene) and pPICZαA plasmids.

[0105] Using the total genomic DNA of Pichia pastoris as a template, primers THI11p2-amp-F and THI11p-R were used to amplify the thiamine-repressed THI11p promoter fragment PpTHI11p-amp;

[0106] The methionine repressible promoter PpMET3p-amp fragment was amplified with primers MET3p-amp-F and MET3p-R.

[0107] pGALS-TcBuster CO V596A Using primers TcBase-thi11p-F and TcBase-aox1tt-R as a template, the DNA fragment TcBase-thi11p / aox1tt encoded by TcBaseCOV596A was amplified from the plasmid DNA.

[0108]Using the pCMV(CAT) T7-SB100 plasmid as a template, the SBase100X coding gene fragment SBase-met3p / aox1tt was amplified with primers SBase-met3p-F and SBase-aox1tt-R.

[0109] Using the pPICZα...

Embodiment 2

[0113] Example 2, Construction of Donor Plasmids pBRAmp-TcBHis and pBRAmp-SBHis

[0114] The pPIC3.5k plasmid and the URA3::actin intron::TcBuster-ClonNat plasmid (obtained from Johns Hopkins University School of Medicine) were extracted.

[0115] Using the pPIC3.5k plasmid as a template, use primers pG-A2-F and Amp-L1-R to amplify the pBR322-Amp fragment with E. coli replication elements, and use His-starg-F and His-starg-R to amplify The HIS4 gene expression cassette fragment His4-arg was obtained.

[0116] URA3::actin intron::TcBuster-ClonNat plasmid was used as a template, and primers TcBL-amp-F / TcBL-arg4-R, TcBR-au-F / TcBR-pbr322-R were used to amplify the two transposable elements of TcBuster respectively. End sequence TcBL-amp / arg fragment and TcBR-pbr322 fragment.

[0117] Plasmid pT2-shp53 / GFP4 (obtained from Addgene) was extracted and used as a template to amplify Sleeping The SBL-amp / arg fragment and the SBR-pbr322 fragment of the two terminal sequences of the bea...

Embodiment 3

[0120] Embodiment 3, the construction of plasmid pTTcBHis4AMazFARS and pMSBHis4AMazFARS

[0121] Using the Pichia pastoris genome as a template, PARS2-aox1tt and PpAOX1p fragments were amplified with primers PARS2-aox1tt-F / PARS2-R and PpAOX1p-F / PpAOX1p-R, respectively.

[0122] Using the genome of Escherichia coli Top10 as a template, the MazF-aox1p / aox1tt fragment with the toxin protein gene mazF was amplified with primers MazF-aox1p-F and MazF-aox1tt-R.

[0123] Using the pPICZα plasmid as a template, the Zeocin-pBR322-pars2 fragment and the AOX1TT fragment were amplified using primers TEF1p-pars2-F / pBR322-R and pG-A1-F / AOX1TT-A1-R, respectively.

[0124] Next, among the several plasmids constructed above, the pBRZeo-TTcBaseCOV596A plasmid is used as a template, and the primers THI11p2-aox1p-F and AOX1TT-A1-R can be used to amplify the PpTHI11p-TcBase-AOX1TT-aox1p fragment containing the TcBase expression cassette . Using the plasmid pBRZeo-MSBase100X as a template, the Pp...

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Abstract

The invention relates to a high-efficiency transposable mutation system and a construction method. By means of the construction method, a double-plasmid transposable system which can gather transposable mutant strains in high efficiency and has low repeatability among the mutant strains is constructed, and a single-plasmid transposable mutation system which can remove transposase encoding gene isconstructed. The transposable mutation system disclosed by the invention can screen and obtain phenotype mutant strains, and then genotypes of the mutant strains can be quickly determined by a chromosome walking technology.

Description

technical field [0001] The invention belongs to the field of molecular biology, and more specifically, the invention relates to an efficient transposition mutation system and a construction method. Background technique [0002] The techniques applied to the random mutation of yeast mainly include chemical mutagenesis and ultraviolet mutagenesis. The genotypes of mutant strains screened by these traditional mutagenesis methods are difficult to identify. The transposon mutation tagging technology is a mutagenesis method that can isolate unknown genes developed according to the characteristics of random insertion of transposons to cause mutations. However, the existing transposon systems used in yeast generally have problems such as low transposition efficiency, low randomness of insertion mutations, and cumbersome operations, which make the transposon mutation system in yeast not widely used. The work of screening mutants and studying the corresponding gene functions has been...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/54C12N1/19
CPCC12N9/1241C12N15/81C12N2830/34C12N2830/36
Inventor 周勉朱锦祥朱巧云张元兴
Owner EAST CHINA UNIV OF SCI & TECH