Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A method for directed evolution of gene promoters

A technology of promoters and bases, applied in the field of directed evolution of gene promoters, can solve the problems of increasing the difficulty of screening, etc., and achieve the effects of low cost, high efficiency and high success rate

Active Publication Date: 2022-02-11
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI +2
View PDF7 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A method for directed evolution of gene promoters
  • A method for directed evolution of gene promoters
  • A method for directed evolution of gene promoters

Examples

Experimental program
Comparison scheme
Effect test

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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a method for directional evolution of gene promoters, the method comprising: using error-prone PCR technology to amplify the promoter to obtain a group of promoter sequences with high mutation frequency; using DNA shuffling technology to remove harmful mutations, Collect beneficial mutations to obtain shuffled promoters; recombinant promoters and galactosidase genes form expression cassettes to transform host cells, and obtain target mutant promoters through blue-white screening and enzyme activity assays. The method of the present invention does not need to analyze the functional region of the gene promoter, is low in cost, efficient and quick, easy to operate, and has a high success rate, and is suitable for the directed evolution of gene promoters in 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com