A kind of genome integration method and application

A genome and integration vector technology, applied in the fields of genetic engineering and biology, can solve problems such as the reduction of editing efficiency, and achieve the effects of simplifying multiple rounds of integration, simple integration methods, and simplifying time-consuming and labor-intensive

Active Publication Date: 2021-11-09
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the upper limit of its integration length is only 10kb. As the length of the integration fragment increases, its editing efficiency will decrease rapidly

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 kind of genome integration method and application
  • A kind of genome integration method and application
  • A kind of genome integration method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Large fragment genome integration technology system construction

[0029] Genome integration technology process, see figure 1 , first construct the integration plasmid containing IS5 sequence, R6K replicon, chloramphenicol resistance gene, target exogenous metabolic pathway, N20 recognition site ( figure 2 ). This system mainly relies on plasmids expressing λ-Red recombinant protein and CRISPR / Cas9 system. With the assistance of the λ-Red recombination protein, the integration plasmid was integrated into the E. coli genome through Campbell-type recombination. Then under the mediation of CRISPR / Cas9, redundant sequences such as IS5 sequence, R6K replicon, and chloramphenicol resistance gene were deleted.

Embodiment 2

[0030] Embodiment 2 different module promoter selection

[0031] 1. Screening of promoters of lycopene synthesis pathway

[0032] This example first uses the pET-30a-trc vector preserved in the laboratory (Su B, Zhang Z, Wu M, Lin J, YangL: Construction of plasmamid-free Escherichia coli for the production of arabitol-free xylitol from corncob hemicellulosic hydrolysate.Sci Rep 2016,6:26567.) as a template, with primers trc-30a-P1 / trc-30a-P2 to amplify the plasmid module, with primers crtI-P1 / crtI-P2, crtE-P1 / crtE-P2, crtB-P1 / crtB-P2 respectively amplifies lycopene synthesis genes crtI, crtE, crtB (crtI, crtE, crtB gene sequence has been disclosed in the patent application number CN 109943492A, the name of the invention: a Chinese patent application for a recombinant yeast strain and its application ), using a recombinant kit to construct the vector pET-trc-IEB. Subsequently, the laboratory preserved pCDFDuet-1 (Su B, Zhang Z, Wu M, Lin J, Yang L: Construction of plasma-free ...

Embodiment 3

[0054] Example 3 Large Fragment Genome Integration

[0055]Using pACYC-phoR (pACYC-phoR-dxs-dxr-yciG-IEB) as a template, primers phoR-dxs-dxr-P1-3, phoR-dxs-dxr-P1-2, phoR-dxs-dxr-P1 / phoR-dxs-dxr-P2 amplifies the phoR-dxs-dxr module; using pACYC-yejG (pACYC-yejG-idi-crtE-yciG-IEB) as a template, using primers yejG-idi-crtE-P1 / yejG-idi- crtE-P2 amplifies the yejg-idi-crtE module; uses pET-trc-IEB as a template, and uses primers IEB-P1 / IEB-P2 to amplify the lacI-trc-IEB module; uses pRC43 (Su B, Zhang Z, Wu M ,Lin J, Yang L:Construction ofplasmid-free Escherichia coli for the production of arabitol-free xylitol from corncob hemicellulosic hydrolysate.Sci Rep 2016,6:26567.) as a template, using primers IS5-R6K-CM-P1 / IS5-R6K -CM-P2, IS5-R6K-CM-P2-2 amplify the IS5-R6K-Cm module, and use the recombination kit to construct the recombinant plasmid pRC-IS5( figure 2 ), the recombinant plasmid pRC-IS5 contains IS5 sequence, R6K replicon, chloramphenicol resistance gene, target exog...

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 genome integration method and application. Specifically disclosed is an Escherichia coli large-segment genome integration vector and its application in synthesizing carotenoids. The E. coli genome integration vector includes a replicon, a target exogenous metabolic pathway, a resistance gene, and an integration site. The integration site is the IS sequence; the engineering bacterium includes Escherichia coli and the above-mentioned integration vector integrated into the genome of Escherichia coli. The present invention uses the IS sequence with a large copy number in the Escherichia coli genome as an integration site to integrate the vector and the genome. Not only the integration method is simple, but also the target gene integrated into the genome is genetically stable, which solves the problem of using the plasmid as an expression vector. When the separation is unstable, it simplifies the existing integration technology, and solves the cumbersome and time-consuming problems of the existing large-segment integration technology.

Description

technical field [0001] The invention belongs to the field of genetic engineering and biotechnology, and relates to a genome integration method and application, in particular to an Escherichia coli large-segment genome integration vector and its application in synthesizing carotenoids. Background technique [0002] Because E. coli genes are easy to operate, there are many commercially available expression vectors, and different promoters and replicons with different copy numbers greatly facilitate the expression of proteins in E. coli; therefore, for the metabolic engineering strategy of E. coli, Currently, plasmids are commonly used as expression vectors. However, the presence of plasmids often leads to excessive metabolic burden, instability of separation, and uncontrollable protein expression; at the same time, in order to maintain the stable presence of plasmids in strains, it is usually necessary to add antibiotics, which not only increases production costs, but also bri...

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): C12N15/70C12N15/90C12N15/65C12N1/21C12P5/02C12R1/19
CPCC12N15/65C12N15/70C12N15/902C12P5/007
Inventor 朱红惠苏卜利宋丹丹冯广达
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap