Agrobacterium homologous recombination system and application thereof

A technology of homologous recombination and recombination system, applied in recombinant DNA technology, virus/phage, introduction of foreign genetic material using vectors, etc. Huge application prospect, high conversion efficiency effect

Pending Publication Date: 2021-12-21
SHANDONG UNIV
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After searching, there is no literature report on the research on the self-ho...

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
  • Agrobacterium homologous recombination system and application thereof
  • Agrobacterium homologous recombination system and application thereof
  • Agrobacterium homologous recombination system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: Construction of serial Agrobacterium homologous recombination expression plasmids

[0070] Apply NCBI's BlastP program to search for RecE / T recombination with Redα / β or Rec prophage of E. Proteins homologous to enzymes, looking for proteins with potential recombinant functions in Agrobacterium. The exonuclease-recombinase operons homologous to RecE / T were found in Agrobacterium tumefaciens str.B6, Rhizobium leguminosarum bv.trifolii WSM597, Rhizobium sp.LC145 and Rhizobium sp.Root483D2 contig_20, respectively, which are ETh1h2h3h4_agroB6 , ETh1h2h3P3_rhi597, ET_rhi145, and ETh_rhi483. Among them, ETh1h2h3h4_agroB6 is about 3410bp long and encodes six proteins, except for two proteins homologous to RecE and RecT, there are 4 hypothetical proteins; ETh1h2h3P3_rhi597 is 3898bp long and encodes five proteins, except for proteins homologous to RecE and RecT, There are also 2 hypothetical proteins and Exo-Pol III; ET_rhi145 is 1394bp long and contains proteins ho...

Embodiment 2

[0076] Embodiment 2: Optimization of electroporation conditions of Agrobacterium tumefaciensC58, Agrobacterium tumefaciensEHA105 and Rhizobiumrhizogenes NBRC 13257

[0077] The optimization of electroporation conditions for Agrobacterium tumefaciensC58 and Agrobacterium tumefaciensEHA105 involves the following two parts:

[0078] (1) Determination of growth curves of Agrobacterium tumefaciensC58 and Agrobacterium tumefaciensEHA105

[0079]First, it is necessary to determine the growth curves of Agrobacterium tumefaciensC58 and Agrobacterium tumefaciensEHA105, so as to determine the optimal time for preparing competent cell culture. Pick three single clones of Agrobacterium tumefaciensC58 and Agrobacterium tumefaciensEHA105, and inoculate them into punctured 2ml EP tubes containing 1.3ml LB liquid medium, culture with shaking at 950rpm for 24 hours at 30°C, draw 100μl seed solution and add it to 900μl liquid LB , mix by pipetting, measure OD 600 . were transferred to 50mlLB ...

Embodiment 3

[0092] Example 3: Comparison of the recombination efficiency of expression plasmids in different combinations of recombination systems in Agrobacterium tumefaciensC58, Agrobacterium tumefaciensEHA105 and Rhizobium rhizogenesNBRC 13257

[0093] In the process of recombination system mining, the applicant connected Redγ derived from Lambda phage or Pluγ derived from Pseudomonas into the Agrobacterium recombination system, and used Red / ET and ccdB reverse screening methods to construct recombinants in different combinations The system expression plasmids are named as follows: pBBR1-kan-P tet -redγ-ETh1h2h3h4_agroB6, pBBR1-kan-P tet -redγ-ETh1h2h3P3_rhi597, pBBR1-kan-P tet -redγ-ET_rhi145, pBBR1-kan-P tet -redγ-ETh_rhi483, pBBR1-kan-P tet -pluγ-ETh1h2h3h4_agroB6, pBBR1-kan-P tet -pluγ-ETh1h2h3P3_rhi597, pBBR1-kan-P tet -pluγ-ET_rhi145 or pBBR1-kan-P tet -pluγ-ETh_rhi483. At the same time, this experiment uses pBBR1-P Rha -gba-kan served as a positive control for recombinat...

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 an agrobacterium homologous recombination system. The agrobacterium homologous recombination system is composed of a series of agrobacterium homologous recombination system expression plasmids which are named as pBBR1-kan-Ptet-ETh1h2h3h4-agroB6 with the nucleotide sequence shown as SEQ ID No.1; pBBR1-kan-Ptet-ETH1h2h3P3-rhi597 with the nucleotide sequence shown as SEQ ID No. 2, pBBR1-kan-Ptet-ET-rhi145 with the nucleotide sequence shown as SEQ ID No. 3, and pBBR1-kan-Ptet-ETh-rhi483 with the nucleotide sequence shown as SEQ ID No. 4. The invention also discloses an application of the recombinant system in agrobacterium tumefaciens mediating homologous recombination of short homologous arms for genome DNA genetic modification and an application in agrobacterium tumefaciens gene knockout. The agrobacterium homologous recombination system can greatly promote genome modification of the agrobacterium, so that genetic modification of the agrobacterium becomes simple and rapid, and the agrobacterium homologous recombination system has huge application prospects in the aspect of constructing agrobacterium strains with high transformation efficiency and host range and in the field of plant genetic engineering.

Description

technical field [0001] The invention relates to a homologous recombination system in Gram-negative bacteria and its construction and application, in particular to an Agrobacterium homologous recombination system and its construction and application, and belongs to the field of microbial genetic engineering. Background technique [0002] The homologous recombination process mediated by RecET and Redαβγ system is collectively referred to as Red / ET homologous recombination engineering. Red / ET homologous recombination engineering can precisely insert, knock out or replace target genes at any position in the E. coli genome. System concatenation can realize the simplification of the E. coli genome. The successful application of Red / ET recombination engineering has promoted the development of the entire genetic engineering technology, in which RecE and RecT homologous recombination proteins are derived from Rac prophage, and Redα, Redβ and Redγ homologous recombination proteins ar...

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): C12N15/74C12N15/65C12N15/66
CPCC12N15/74C12N15/65C12N15/66C12N2800/101C12N2800/30C12N2800/60C12N2800/70
Inventor 符军李瑞娟边志龙李珊珊杨润雨涂强张友明
Owner SHANDONG UNIV
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