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Engineering bacterium for degrading polycyclic aromatic hydrocarbon, engineering reforming method and application thereof

A technology of polycyclic aromatic hydrocarbons and engineering bacteria, which is applied in the fields of genetic engineering and biodegradation, can solve the problems of difficult conversion of Pseudomonas bacteria and low conversion rate of heat shock, and achieve good degradation ability and improve the effect of degradation ability

Active Publication Date: 2019-01-11
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the transformation of Pseudomonas is difficult, and the conversion rate of heat shock is low. In order to obtain high transformation efficiency, more researchers tend to use conjugation or electric shock transformation to introduce plasmids into recipient bacteria.

Method used

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  • Engineering bacterium for degrading polycyclic aromatic hydrocarbon, engineering reforming method and application thereof
  • Engineering bacterium for degrading polycyclic aromatic hydrocarbon, engineering reforming method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Clone catA2 gene from Pseudomonas aeruginosa SA1

[0029] (1) Extract the whole genome and plasmid of Pseudomonas aeruginosa SA1;

[0030] (2) According to the degradation gene sequence encoding polycyclic aromatic hydrocarbon ring lyase, design specific primers to specifically primer catAF and catAR.

[0031] catAF: 5'-ATGACCGTGAAGATATCTCACAC-3'

[0032] catAR: 5'-TTAGATTTCTTGCAAGGCTCTCG-3'

[0033] Using the genome and plasmid as a template, amplify a specific band of about 900bp, such as figure 2 As shown, the size corresponds to the size of the catA2 gene;

[0034] (3) Send the amplified product to Beijing Jinweizhi Biotechnology Co., Ltd. for sequencing, and the sequencing result is shown in SEQ ID No.1. Comparing the sequencing results with the catA2 gene encoding polycyclic aromatic hydrocarbon ring lyase, the results show that the two sequences are completely consistent, and it can be determined that the catA2 gene has been cloned from the bacteri...

Embodiment 2

[0035] Example 2 Construction of recombinant expression vector pBBR1MCS-5-oprL-catA2

[0036] (1) For the degradation gene catA2, add HindⅢ and XbaⅠ restriction sites at both ends of the fragment by PCR method, and use FastDigest endonucleases HindⅢ and XbaⅠ to perform digestion. The reaction system is: 5 μL 10*FDbuffer, 2.5 μL HindⅢ, 2.5 μL XbaⅠ, 30 μL catA2 gene with restriction enzyme site and 10 μL ultrapure water. The reaction conditions are: 37°C, 1h. Similarly, the pBBR1MCS-5 plasmid was digested with FastDigest endonucleases HindⅢ and XbaI, purified and recovered using the PCR purification kit. The digested catA2 gene and pBBR1MCS-5 plasmid were ligated. The reaction system is: 1 μL 10*T4DNALigase Buffer, 1 μL T4DNA Ligase, 6 μL digested nucleotide fragments and 2 μL digested pRSFDuet plasmid. The reaction conditions are: 22°C, 10 min. After enzyme digestion and ligation, the competent cells E.coli DH5α were transformed, the positive clones were screened by colony ...

Embodiment 3

[0038] Embodiment 3 recombinant vector is transformed in Pseudomonas aeruginosa SA1

[0039] The detailed construction steps of recombinant expression vector pBBR1MCS-5-oprL-catA2 transformed into Pseudomonas aeruginosa chassis strain SA1 are as follows:

[0040] (1) Add 2.5 μL of the recombinant expression vector pBBR1MCS-5-oprL-catA2 to 50 μL of Pseudomonas aeruginosa SA1 competent cells, and gently pipette to mix;

[0041] (2) Put the mixture into a 2mm electric cup and shock at 2.5KV for 5-6ms. Then quickly add 1mL anti-resistant LB medium, recover at 37°C, 200rpm for 2h, spread gentamicin-resistant LB solid medium plate, and culture overnight at 37°C;

[0042] (3) The positive transformants verified by colony PCR were selected and cultured overnight in 5 mL LB medium to obtain engineering Pseudomonas aeruginosa PH2, and the bacterial strain was preserved.

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Abstract

The invention relates to an engineering bacterium for degrading polycyclic aromatic hydrocarbon, an engineering reforming method and an application thereof. The engineering reforming method comprisesthe following steps: cloning from pseudomonas aeruginosa SA1, thereby acquiring a degrading gene, namely, catA2 gene, for encoding ring splitting decomposition of dioxygenase, wherein the nucleotide sequence is shown as SEQ ID No.1 and the nucleotide sequence of specific oprL promoter is shown as SEQ ID No.2; selecting a broad host range plasmid pBBR1MCS-5 as a carrier; respectively cloning catA2gene and oprL promoter into the pBBR1MCS-5 carrier, shifting to a recipient cell for clonal expansion and screening positive clone, thereby acquiring a target recombinant vector pBBR1MCS-5-oprL-catA2;utilizing pseudomonas aeruginosa SA1 as a host to convert the recombinant vector pBBR1MCS-5-oprL-catA2 into a pseudomonas aeruginosa strain through an electric shock method, thereby constructing engineering pseudomonas aeruginosa. The engineering bacterium has the capacity of degrading polycyclic aromatic hydrocarbon.

Description

technical field [0001] The invention relates to a polycyclic aromatic hydrocarbon degrading engineering bacterium and its engineering transformation method and application, belonging to the fields of biodegradation and genetic engineering. Background technique [0002] Polycyclic aromatic hydrocarbons refer to hydrocarbons containing two or more benzene rings in their molecules, such as naphthalene, anthracene, phenanthrene, pyrene, biphenyl, terphenyl, etc. Polycyclic aromatic hydrocarbons widely exist in soil, water and air, have poor water solubility and thermal stability, and a considerable part of them have chronic toxicity, carcinogenicity, teratogenicity and mutagenicity, which are harmful to human health and ecology. The environment has caused great harm, and it is a class of dangerous compounds in the environment that need to be studied. The fate of polycyclic aromatic hydrocarbons in the environment includes volatilization, photooxidation, chemical oxidation, bioa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/78A62D3/02C12R1/385A62D101/20
CPCA62D3/02A62D2101/20C12N9/0069C12N15/78
Inventor 贾晓强贺赟姜大伟
Owner TIANJIN UNIV
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