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Method for carrying out directional evolution on gene promoter

A directional evolution, promoter technology, applied in DNA preparation, DNA/RNA fragment, recombinant DNA technology, etc., can solve the problem of increasing the difficulty of screening, and achieve high efficiency and protein yield, simple operation and high success rate. Effect

Active Publication Date: 2017-11-10
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI +2
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Problems solved by technology

However, if the wild-type promoter sequence is not mutated or amplified by a method with a low mutation rate, the recombinant promoter after DNA shuffling contains few beneficial mutations, resulting in most of the library being wild-type populations, greatly increasing Difficulty of screening

Method used

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  • Method for carrying out directional evolution on gene promoter
  • Method for carrying out directional evolution on gene promoter
  • Method for carrying out directional evolution on gene promoter

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Embodiment 1

[0034] The present invention introduces point mutations into the tac promoter of Escherichia coli at a mutation frequency of 1%-2% through error-prone PCR technology, then degrades the promoter sequence with DNase I and recovers small fragments, and obtains recombination through Primerless PCR and Primer PCR The mutant tac promoter was cloned into the expression vector with galactosidase reporter gene, transformed into Escherichia coli, and the promoter mutation library was obtained. Spread the library on an LB plate containing 80 mg / ml X-gal, extract the clone with the deepest blue color and expand it for culture, and perform enzyme activity assay of β-galactosidase and clone sequencing analysis of shuffled tac promoter, respectively.

[0035] (1) Reorganization of the tac promoter

[0036] Error-prone PCR amplification of the tac promoter. Due to Mg 2+ Can stabilize non-complementary base pairs during PCR; Mn 2+ The specificity of the polymerase to the template can be red...

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Abstract

The invention provides a method for carrying out directional evolution on a gene promoter. The method comprises the following steps: amplifying the promoter by adopting an error-prone PCR technology, thus obtaining a group of promoter sequences with high mutation frequency; removing harmful mutation by adopting a DNA shuffling technology, and collecting beneficial mutation, thus obtaining the promoter after shuffling; forming an expression cassette by adopting the recombinant promoter and a galactosidase gene, transforming host cells by adopting the expression cassette, and carrying out blue and white spot screening and enzyme activity determination, thus obtaining the target mutation promoter. With the method provided by the invention, the functional region of the gene promoter does not need to be analyzed, the cost is low, the operation is efficient, rapid, easy and convenient, the success rate is high, and the method is suitable for carrying out directional evolution on gene promoters of escherichia coli or other bacteria, fungi and mammalian cells.

Description

technical field [0001] The invention relates to a method for directional evolution of gene promoters, in particular to a method for directional evolution of Escherichia coli gene promoters by using DNA shuffling technology combined with error-prone PCR technology. Background technique [0002] Promoter is a DNA sequence that RNA polymerase specifically recognizes and binds to. It is an integral part of a gene and controls the initiation time and degree of gene expression (transcription). Prokaryotic promoters have three important regions: the -10 sequence, the -35 sequence and the nucleotides in between. The -10 sequence is the TATA box, which controls the initiation of transcription, the -35 sequence binds RNA polymerase during transcription, and the change in the number of nucleotides between the -10 region and the -35 region will also affect the gene transcription activity. Engineering these control elements can alter the efficiency and properties of the promoter. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/113
CPCC12N15/102C12N15/113
Inventor 万晓春李俊鑫刘绿艳吴高慧
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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