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Recombinant plasmid and application of same in identification of gene expression of thiobacillus aeruginosa

A technology of Thiobacillus thermophiles and recombinant plasmids, applied in the field of genetic engineering

Pending Publication Date: 2019-09-20
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After searching, there is no report on the recombinant plasmid used to identify the gene expression intensity in acidophilus thermophilic Thiobacillus and its application in the identification of gene expression in acidophilic autotrophs

Method used

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  • Recombinant plasmid and application of same in identification of gene expression of thiobacillus aeruginosa
  • Recombinant plasmid and application of same in identification of gene expression of thiobacillus aeruginosa
  • Recombinant plasmid and application of same in identification of gene expression of thiobacillus aeruginosa

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Effect test

Embodiment 1

[0031] Example 1 The recombinant plasmid pSKL-P of the reporter gene containing the tetH promoter tetH - Construction of Luc-Chl

[0032](1) Genome extraction: Genome extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract the genome of wild-type A. caldus bacteria for future use.

[0033] (2) Plasmid extraction: Plasmids pJRD215 and pSKL were extracted using OMEGA plasmid extraction kit (purchased from Qingdao Keshengran Co., Ltd.) for future use.

[0034] (3) Construction of firefly luciferase gene (luc) and chloramphenicol resistance gene (cat) cluster: The original sequences of firefly luciferase gene (luc) and chloramphenicol resistance gene (cat) were submitted to Suzhou Golden Weizhi Biotechnology Co., Ltd. carried out sequence optimization to construct a cluster and construct it on the plasmid pET22b, and the recombinant plasmid was named pET22b-Luc-Chl.

[0035] (4) Recombinant plasmid pSKL-P tetH -Construction of Luc-...

Embodiment 2

[0052] Embodiment 2 Recombinant bacteria A.caldus (pSKL-P tetH - Construction of Luc-Chl)

[0053] The positive cloning plasmid with correct sequencing, that is, the recombinant plasmid pSKL-P of the reporter gene containing the tetH promoter tetH -Luc-Chl transformed Escherichia coli SM10, and the Escherichia coli SM10 containing the recombinant plasmid was used as the donor bacterium, and the wild type A. caldus was used as the recipient bacterium, and the recombinant plasmid was transferred by conjugation Transferred into acidophilic Thiobacillus acidophilus, using chloramphenicol as a selection marker to obtain the recombinant strain of acidophilic Thiobacillus, named recombinant bacteria A.caldus (pSKL-P tetH -Luc-Chl)

[0054] During the above-mentioned conjugation transfer process, the donor bacteria and the recipient bacteria were shaken to the mid-logarithmic phase. In order to ensure that the pili of the donor and recipient bacteria were not damaged, the speed of t...

Embodiment 3

[0055] Embodiment 3 Recombinant bacteria A.caldus (pSKL-P tetH Definition and identification of -Luc-Chl) gene expression intensity

[0056] 1. Bacteria collection. The bacterial solution was allowed to stand at room temperature for 3 minutes, and the bacterial solution was filtered with filter paper to remove sulfur powder. Centrifuge at 10000g at 4°C for 6min to collect the cells. Pour off the waste liquid, add 1ml PBS buffer (10mM PO 4 3- , 0.8% NaCl), wash the bacteria, transfer to a 1.5ml EP tube, centrifuge at 3000g for 30s.

[0057] 2. Bacteria washing and lysing. Aspirate the bacteria, transfer to a 10ml centrifuge tube, add 5ml PBS, vortex and mix, centrifuge at 10000g, 4°C for 1min, discard the supernatant, and repeat the above process. Add appropriate amount of PBS to adjust the cell size to 2×10 8 Cells were collected by centrifugation at 10000g for 5min at 4°C, and the supernatant was discarded. Centrifuge again for 15 s, absorb the residual liquid, add 20...

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Abstract

The invention discloses a recombinant plasmid containing a tetH promoter, a firefly luciferase gene luc, a chloramphenicol resistance gene cat, a replicator oriV, a junction transfer gene mob, replication protein genes repA, repB and repC and a Kanamycin resistance gene Kanr element, and the nucleotide sequence is shown in SEQ ID No.1. The invention further discloses an application of the recombinant plasmid in identification of gene expression of thiobacillus aeruginosa, luciferase activity of recombinant bacteria is detected after establishment, and the intensity of gene expression of thiobacillus aeruginosa is determined according to the size of the fluorescence value. The method has the advantages of low background, high sensitivity, simple operation, high repeatability and accuracy; establishment of engineering bacteria with stress resistance and high efficiency leaching, for realizing the screening of the efficient promoters and the identification of the gene expression intensity in the thiobacillus autotrophic bacteria has the theoretical guiding significance and the practical application value.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a recombinant plasmid and its application in identification of acidophilic thermophilic thiobacterium gene expression. Background technique [0002] At present, our country is facing issues such as resource scarcity and climate rise. In order to successfully achieve the sustainable development goals and ensure the ecological sustainability of the global mineral supply, metal recycling and technological changes can help maintain the supply for industrial use. In the foreseeable future, a large amount of minerals still need to be mined for industrial use. Traditional mining methods have more and more problems such as high ore grade requirements, high cost, high energy consumption, and heavy pollution. Therefore, it is urgent to develop and use new technologies and processes to expand ore sources, improve ore utilization, reduce energy consumption, and reduce environme...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/65C12Q1/66
CPCC12N15/63C12N15/65C12Q1/66
Inventor 陈林旭陈显轲高玉辉林建群林建强
Owner SHANDONG UNIV
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