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

Mutator strain with loss of correcting function of escherichia coli dnaQ gene, and preparation method and applications thereof

A technology of Escherichia coli and gene correction, applied in the field of biotechnology engineering, to reduce working time cost and improve efficiency

Active Publication Date: 2019-04-02
WUHAN INST OF BIOENG
View PDF2 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The prior art has not yet used dnaQ gene mutated strains as mutator strains for biological mutagenesis

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
  • Mutator strain with loss of correcting function of escherichia coli dnaQ gene, and preparation method and applications thereof
  • Mutator strain with loss of correcting function of escherichia coli dnaQ gene, and preparation method and applications thereof
  • Mutator strain with loss of correcting function of escherichia coli dnaQ gene, and preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The features and advantages of the present invention can be further understood through the following detailed description in conjunction with the accompanying drawings. The examples provided are only illustrative of the method of the present invention and do not limit the rest of the present disclosure in any way. [Example 1] The gene dnaQ encoding Escherichia coli DNA polymerase IIIε subunit was knocked out using the λ-Red recombinase method

[0036] (1) Design primers:

[0037] The primers used for homologous recombination are dnaQtetR and dnaQtetA; the design principle is that the 5' end is the homologous region of the dnaQ gene (not underlined), and the 3' end is the homologous sequence of the tetracycline resistance gene tetRA (underlined), recombination The primers used for body detection were EdnaQU and EdnaQD. The sequences of these primers are shown in Table 1 and were synthesized by Beijing Aoke Dingsheng Biotechnology Co., Ltd.

[0038] Table 1 The primers...

Embodiment 2

[0046] [Example 2] Functional analysis of dnaQ mutants

[0047] Pick the wild-type DH5α and [Example 1] the dnaQ mutant identified by PCR with EdnaQU and EdnaQD primers and culture them in 5mL LB medium at 200r / min at 37°C overnight, and then collect the cells by centrifugation, concentrate to 1ml, and gradient Dilute, then take 100 μl and spread on LB solid plate and LB solid plate containing nalidixic acid (the concentration of nalidixic acid is 30ug / ml), after culturing at 37°C for 18 hours, observe the growth of the colonies and record the number of colonies on each plate (CFU). The ratio of the number of colonies on the nalidixic acid plate to the number of colonies on the LB plate was used as the mutation rate of resistance to nalidixic acid, and each sample was repeated three times.

[0048] The wild-type DH5α and the dnaQ mutant of [Example 1] were shaken and cultured in LB medium at 37°C overnight. After the overnight culture solution was serially diluted, the same ...

Embodiment 3

[0054] [Example 3] Mutation test of serine receptor protein gene tsr

[0055] The plasmid pJC3 containing the serine receptor protein gene tsr was transformed into the competent cells of the dnaQ mutant of [Example 1] by heat shock method, plated on an LB plate containing 100ug / ml ampicillin, and overnight at 37°C to cultivate. Pick different colonies and inoculate them into 1ml of LB liquid medium respectively. After overnight shaking culture at 37°C, take 10ul of the culture solution and spread it on a plate containing 30ug / ml nalidixic acid, and culture it overnight at 37°C. Plasmids were extracted from the original colony that produced the largest number of resistant colonies on the nalidixic acid plate, and used as a tsr mutation library.

[0056] The mutant library plasmid was transformed into strain uu2610 (△MCPs△CheRCheB) whose methyl modification system CheR, CheB and all chemotactic receptors were knocked out by heat shock method. Streak the transformed cells on a ...

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 discloses a mutator strain with loss of correcting function of escherichia coli dnaQ gene, and a preparation method and applications thereof. The mutator strain is obtained by knocking out, inserting inactivating or mutating gene dnaQ of encoded escherichia coli DNA polymerase III epsilon subunit so as to lose the correcting function during DNA replication. The method knocks out thegene dnaQ of eth encoded escherichia coli DNA polymerase III epsilon subunit by using a lambada-Red recombinase method, and obtains the mutator strain with loss of correcting function during DNA replication. The mutation capacity of the strain is 25000 times as high as that of wild types, and the strain is an efficient means of gene mutagenesis. At the same time, the strain reduces the working time cost for screening mutants with excellent traits, and improves the efficiency of scientific research work and engineering bacteria selection.

Description

technical field [0001] The invention belongs to the field of biotechnology engineering, and in particular relates to a gene mutator strain with an increased mutation rate of DNA replication caused by knockout, insertion inactivation, or loss of its correction function due to mutation of the Escherichia coli danQ gene; and the mutator The strain is used to mutate the target gene to achieve the purpose of screening excellent traits and the strain is directly used for breeding mutants with specific traits. Background technique [0002] The mutation frequency of microbial strains under natural conditions is low, and the mutation range is not large, and the gene structure is generally difficult to change. Therefore, in order to obtain strains with specific traits in production and research, it is often necessary to carry out mutation breeding on microorganisms . Microbial mutation breeding refers to the method of using physical, chemical or biological factors to induce mutations...

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
IPC IPC(8): C12N1/21C12N15/70C12N15/90C12R1/19
CPCC12N9/1252C12N15/70C12N15/902C12Y207/07007
Inventor 王松太郭书奎周鑫鑫
Owner WUHAN INST OF BIOENG
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