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Genome integration method and application

A genome and integrated carrier technology, applied in the field of genetic engineering and biology, can solve the problems of reduced editing efficiency and achieve the effect of simple integration method, simplified time-consuming and labor-intensive, and stable genetics

Active Publication Date: 2020-06-30
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • 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

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  • Genome integration method and application
  • Genome integration method and application
  • 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 integrated 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...

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Abstract

The invention discloses a genome integration method and an application. Specifically, the invention discloses an escherichia coli large fragment genomic integration vector and an application of the vector in synthesis of carotenoids. The escherichia coli genomic integration vector includes a replicon, a target exogenous metabolic pathway, a resistance gene and an integration site, wherein the integration site is an IS sequence; and engineering bacteria include escherichia coli and the above integration vector integrated into the escherichia coli genome. The method uses the IS sequence with a large number of copies in the escherichia coli genome as the integration site to integrate the vector with the genome, not only the integration method is simple, but also the genetic integration of thetarget gene integrated into the genome is stable, so that the problem of unstable separation when plasmid is used as an expression vector is solved, a current integration technology is simplified, and the problem of cumbersome and time-consuming operation of the current large fragment integration technology is solved.

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

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Application Information

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Patent Type & Authority Applications(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
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