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Novel recombination system in pseudomonas and application thereof

A recombination system, Pseudomonas technology, applied in the field of microbial genetic engineering, can solve the problems of restricting the research and transformation of Pseudomonas genome, low efficiency, and low efficiency

Active Publication Date: 2017-07-07
RECOM QINGDAO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional gene manipulation technology uses techniques such as restriction endonuclease, DNA ligase and PCR amplification to realize the modification of the target gene in vitro, such as insertion, knockout and point mutation, but the operation is cumbersome and the efficiency is low. The limitation of cutting sites, cumbersome operation and low efficiency limit the research and transformation of Pseudomonas genome
There are few reports on the research and patent of the recombinant system in Pseudomonas (Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas fluorescens and Pseudomonas putida) and its construction and application

Method used

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  • Novel recombination system in pseudomonas and application thereof
  • Novel recombination system in pseudomonas and application thereof
  • Novel recombination system in pseudomonas and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0053] Preparation of electroporated cells for recombination:

[0054] Various expression plasmids were transformed into Escherichia coli GB05 from GeneBridges in Germany, Pseudomonas aeruginosa PAO1 from DSMZ in Germany (Accession No.: DSM 1707), Pseudomonas fluorescens pf5 from American Type Culture Collection ATCC (Accession No.: BAA-477 TM ), Pseudomonas syringae DC3000 were purchased from the American Type Culture Collection ATCC (preservation number: BAA-871 TM ), Pseudomonas putida KT2440 were purchased from DSMZ, Germany (Accession No.: DSM6125). Escherichia coli competent cells were prepared according to the procedures reported in the previous literature. Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas syringae, Pseudomonas putida, and strains with expression plasmids were first cultured overnight and then diluted to 1.3 ml of LB medium (Pseudomonas syringae In KB culture medium), the initial OD 600 The value of is about 0.085. Cultivate accordin...

Embodiment 1

[0055] Example 1: Construction of a series of homologous recombination expression plasmids

[0056] Using the amino acid sequence of λBeta or rac RecT as a reference, BLAST analyzes the genomes from Pseudomonas and Pseudomonas phages, looking for proteins with potential recombination functions. Two operons containing paired exonuclease-recombinase were found from Pseudomonas and Pseudomonas phage, one operon is RecTEpsy from Pseudomonas syringae, which has been successfully used in Construction of recombinant system; the second operon comes from Pseudomonas phage vB_PaeP_Tr60_Ab31, this operon encodes three proteins: Orf38 (B) contains 177 amino acids, and the sequence has 62% homology with Redβ; Orf37 (A) It contains 195 amino acids, and its sequence has 32% homology with Redα; Orf36(S) contains 148 amino acids, and its sequence has 54% homology with the single-stranded DNA binding protein SSB of Escherichia coli.

[0057] A series of recombinant functional plasmids are cons...

Embodiment 2

[0061] Example 2: Efficiency Comparison of Different Recombination Systems in Pseudomonas

[0062] pBBR1-Rha-red_gba-Kan, pBBR1-Rha-plu_gba-kan, pBBR1-Rha-TEpsy-kan, pBBR1-Rha-pluGam-TEpsy-kan, pBBR1-Rha-redGam-TEpsy-kan, pBBR1-Rha-BAS -kan, pBBR1-Rha-pluGam-BAS-kan, pBBR1-Rha-RedGam-BAS-kan were transformed into four kinds of Pseudomonas (Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas fluorescens) bacteria and Pseudomonas putida). figure 1 A is a schematic diagram of the comparison of recombinant system efficiencies. Pseudomonas aeruginosa grows well at both 37°C and 30°C, so a comparison of efficiencies at these two temperatures is needed. Pseudomonas aeruginosa itself has multiple drug resistances and it is easy to obtain stronger drug resistance mechanisms, so the correct rate should be calculated after comparing the recombination efficiency in Pseudomonas aeruginosa.

[0063] In Pseudomonas aeruginosa, the addition of Redγ and Pluγ proteins to the BAS recomb...

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Abstract

The invention discloses a novel recombination system in pseudomonas. The recombination system is established according to lambda Red-like operons (Orf38, Orf37 and Orf36) from pseudomonas aeruginosa phage Ab31 and RecET-like operon (recTEPsy) from pseudomonas syringae pv.syringae B728a prophage. According to the recombination system, apart from two genes of host specific exonuclease and single-stranded DNA annealing protein, a third gene is exonuclease recBCD profilin, and a third gene or a fourth gene also can be single-stranded DNA binding protein SSB from bacterial chromosome, bacterial plasmid or other sources. Function test results show that a hybrid system composed of the lambda Red-like operons and the RecET-like operon and Red gamma / Plu gamma can operate in pseudomonas and the SSB protein can remarkably enhance the recombination efficiency. Those combination systems can be utilized for efficient and succinct plasmid modification and point mutation and knock-in and knock-out genome modification, and have a wide application prospect.

Description

technical field [0001] The present invention relates to a kind of recombinant system in Gram-negative bacilli and its construction and application, especially a kind of Pseudomonas (Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas fluorescens or / and Pseudomonas putida The invention relates to a recombinant system and its construction and application in the single cell bacteria), which belong to the field of microbial genetic engineering. Background technique [0002] Phage-encoded recombination systems can be widely used in modifying prokaryotic genomes and constructing eukaryotic transgenic species. It only needs 35 nucleotides as a homology arm, and its applications mainly include the construction of mutants - point mutations, deletions, insertions and substitutions. Shorter homology arms are the main advantage of this technique, which is named recombination engineering. [0003] In E. coli, the most widely used recombination systems are the λRed system and the ...

Claims

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Application Information

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IPC IPC(8): C12N15/78
CPCC12N15/78
Inventor 张友明尹佳符军郑文韬
Owner RECOM QINGDAO BIOTECH CO LTD
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