Burkholderia cenocepacia homologous recombination system and application thereof

A Burkholderia, homologous recombination technology, applied in the direction of recombinant DNA technology, virus/bacteriophage, using vectors to introduce foreign genetic material, etc., can solve the problems of lack of efficient and simple genetic manipulation methods

Active Publication Date: 2021-02-26
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Burkholderia lacks efficient and simple genetic manipulation methods, so it is necessary to establish an efficient and simple genetic operating system in Burkholderia
After searching, there is no report on the recombinant system of Burkholderia and its construction and application

Method used

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  • Burkholderia cenocepacia homologous recombination system and application thereof
  • Burkholderia cenocepacia homologous recombination system and application thereof
  • Burkholderia cenocepacia homologous recombination system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Construction of a series of Burkholderia homologous recombination expression plasmids

[0071] Using the amino acid sequence of λBeta or rac RecT as a reference, BLAST analyzes the genomes of Burkholderia and its phages to find potential proteins with recombinant functions. The exonuclease-recombinase operons homologous to RecET were found in Burkholderia sp.BDU8, Burkholderia sp.TJI49, and Burkholderia sp.YI23, which are EThe_bdu8, ETh_tji49, and ETh1h2e_yi23, respectively. Among them, EThe_bdu8 encodes four proteins: E_bdu8 contains 321 amino acids, its sequence has 34% homology with RecE, T_bdu8 contains 320 amino acids, its sequence has 36% homology with RecT, h_bdu8 and e_bdu8 are hypothetical proteins; ETh_tji49 encodes three proteins: E_tji49 contains 217 amino acids, its sequence has 24% homology with RecE, T_tji49 contains 339 amino acids, its sequence has 34% homology with RecT, h_tji49 is a hypothetical protein; ETh1h2e_yi23 encodes five Protein: ...

Embodiment 2

[0077] Example 2: Optimization of Burkholderia glumae PG1 electroporation conditions

[0078] The optimization of Burkholderia glumae PG1 electroporation conditions involves the following four parts:

[0079] (1) Determination of the growth curve of Burkholderia glumae PG1

[0080] First, the growth curve of Burkholderia glumae PG1 needs to be measured to determine the optimal time for preparing competent cell culture. Pick three Burkholderia glumae PG1 monoclonal clones, inoculate them into 2ml EP tubes containing 1.8ml LB liquid medium, 30°C, 950rpm shaking culture for 18-24h, pipette 100μl seed solution into 900μl liquid LB, Mix by pipetting and measure OD 600 . Draw an appropriate amount of seed liquid and inoculate it into 50ml LB liquid medium containing kanamycin (5 μg / ml), so that the initial OD 600 =0.1, 30°C, 200rpm shaking culture, every 2 hours, draw 1ml of bacterial solution to measure OD 600 , according to the OD at each time point 600 Draw the growth curve...

Embodiment 3

[0089] Example 3: Detection of recombination function of recombinant system expression plasmids in Burkholderia glumae PG1 and E.coli GB2005

[0090] The recombinant system expression plasmids pBBR1-Rha-redgba-kan, pBBR1-Rha-BA_7029-kan, pBBR1-Rha-EThe_bdu8-kan, pBBR1-Rha-ETh_tji49-kan, pBBR1-Rha-ETh1h2e_yi23-kan, pBBR1- Rha-TEpsy-kan, pBBR1-Rha-BAS-kan and pBBR1-Rha-GBAS-kan were electrotransformed into Burkholderia glumae PG1 and E.coli GB2005, respectively.

[0091] In Burkholderia glumae PG1, pBBR1-apra-kan is used as a template, the nucleotide sequence of which is shown in SEQID No.4, primer-PG1-chrom1C4-Papra-insert-5-80 and primer-PG1-chrom1C4-Papra-insert -3-80 is a primer for amplifying apraR, and the two ends of the PCR product apraR-LCG have homology arms with the front sequence of C1BGC4 in the Burkholderia glumaePG1 genome, and the purified apraR-LCG is inserted into the front of the gene BGL_RS05915 in the Burkholderia glumaePG1 genome, and carried out Wire loop...

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Abstract

The invention discloses a Burkholderia cenocepacia homologous recombination system, which consists of one series of Burkholderia cenocepacia homologous recombination system expression plasmids which are independently called after pBBR1-Rha-EThe_bdu8-kan of which the nucleotide sequence is disclosed by SEQ ID NO.1, pBBR1-Rha-ETh_tji49-kan of which the nucleotide sequence is disclosed by SEQ ID NO.2, and pBBR1-Rha-Eth1h2e_yi23-kan of which the nucleotide sequence is disclosed by SEQ ID NO.3. The invention also discloses application of the recombination system in carrying out genome DNA genetic modification on the homologous recombination of a mediated short homologous arm in the Burkholderia cenocepacia, and application of the recombination system in carrying out gene insertion in the Burkholderia cenocepacia to activate a silent gene cluster. The Burkholderia cenocepacia homologous recombination system disclosed by the invention can greatly accelerate modification of the genome of the Burkholderia cenocepacia, so that the genetic modification of the Burkholderia cenocepacia becomes simple and quick, an important tool is provided for further mining a secondary metabolite in the Burkholderia cenocepacia, and the Burkholderia cenocepacia homologous recombination system has a wide application prospect.

Description

technical field [0001] The invention relates to a homologous recombination system in Gram-negative bacteria and its construction and application, in particular to a homologous recombination system in Burkholderia and its construction and application, belonging to the field of microbial genetic engineering. Background technique [0002] The Red / ET homologous recombination technology can be used to precisely modify the genome of E. coli. The RecET homologous recombination protein is derived from the E. coli Rac prophage, and the Redαβγ homologous recombination protein is derived from the Red operon of the E. coli λ phage. RecET and Redαβγ homologous recombination technologies have greatly improved the recombination efficiency in Escherichia coli and provided an effective method for its genetic modification. Among them, RecE and Redα have 5’–3’ exonuclease activity, and RecT and Redβ are DNA single-strand annealing proteins. [0003] Redγ inhibits the exonuclease activity of R...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/90C12N15/66C12N15/65
CPCC12N15/74C12N15/902C12N15/66C12N15/65C12N2830/002Y02A50/30
Inventor符军李瑞娟史洪波赵晓雨张友明
OwnerSHANDONG UNIV