A marker-free gene knockout method for extreme acidophilic Thiobacillus ferrooxidans

A technology of Thiobacillus ferrooxidans and marker-free genes, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problem of antibiotic resistance gene residues, less antibiotic usability, unstable plasmid vectors, etc. question

Active Publication Date: 2011-11-30
SHANDONG UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

[0005] Aiming at the instability of the plasmid vector in A.ferrooxidans above, the usability of fewer types of antibiotics, the residue of antibiotic resistance genes in the existing knockout method, and the inconvenience of carrying out multi-gene knockout research, the present invention provides a A marker-free gene knockout method for A.ferrooxidans based on the principle of homologous recombination

Method used

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  • A marker-free gene knockout method for extreme acidophilic Thiobacillus ferrooxidans
  • A marker-free gene knockout method for extreme acidophilic Thiobacillus ferrooxidans
  • A marker-free gene knockout method for extreme acidophilic Thiobacillus ferrooxidans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Unmarked knockout of the extreme acidophilic Thiobacillus ferrooxidans phosphofructokinase gene (pfkB, AFE_1807 gene)

[0041] (1) Construction of a suicide plasmid for knocking out the A.ferrooxidans ATCC 23270AFE_1807 gene

[0042] 1. Construction of the initial plasmid for A.ferrooxidans gene knockout

[0043] Primers were designed according to the sequence information of plasmid pSW29T published in GenBank: AY733073.1:

[0044] 29TKpnI:

[0045] 5′-CGGC GGTACCTAGGGATAACAGGGTAAT AGCGCTTTTCCGCTGCATAACCC-3′

[0046] 29TSalI:

[0047] 5′-CTAA GTCGACTGATCAACGCGTCTCGAG GCCGGCCAGCCTCGCAGAGCAGG-3′

[0048] The 29TKpnI and 29TSalI primers use the plasmid pSW29T as a template to amplify in vitro the fragment with the oriT region by PCR (polymerase chain reaction). The I-SceI recognition and action site sequence of 18 bp is added inside the Kpn I site of the upstream primer, and the Bcl I, Mlu I and Xho I recognition site sequences are added inside the Sal ...

Embodiment 2

[0117] Example 2: Unmarked knockout of the extreme acidophilic Thiobacillus ferrooxidans tetrathionite hydrolase gene (tetH, AFE_0029 gene)

[0118] (1) Construction of a suicide plasmid for knocking out the A. ferrooxidans ATCC 23270 AFE_0029 gene

[0119] 1. construction is used for the initial plasmid of A.ferrooxidans gene knockout (same as embodiment 1, omitted)

[0120] 2. Construction of a suicide plasmid for knocking out the AFE_0029 gene

[0121] Primers were designed according to the AFE_0029 gene and its upstream and downstream gene sequences in the genome sequence of A. ferrooxidans standard strain ATCC 23270 published in GenBank: CP001212.1:

[0122] HA1F:

[0123] 5′-GCT GAATTC CTCGTCCATCATTTTTTTGCGG-3′

[0124] HA1R:

[0125] 5′-TACAAGCTTC TCTAGA CAGATGGTGCGTTTTCC-3'

[0126] HA2F:

[0127] 5′-CGCGAATTC TCTAGA CCGTCTACCTGACCAACTC-3′

[0128] HA2R:

[0129] 5′-GCTAAGCTT GAGCTC CGTCACGTCACTGCGAAAT-3′

[0130] HA1F and HA1R primers, HA2F and HA2R p...

Embodiment 3

[0169] Example 3: Unmarked knockout of the extreme acidophilic Thiobacillus ferrooxidans metallo-β-lactamase gene (AFE 0269 gene, referred to as sdo)

[0170] (1) Construction of a suicide plasmid for knocking out the A.ferrooxidans ATCC 23270 AFE_0269 gene

[0171] 1. construction is used for the initial plasmid of A.ferrooxidans gene knockout (same as embodiment 1, omitted)

[0172] 2. Construction of a suicide plasmid for knocking out the AFE_0269 gene

[0173] Design primers according to the AFE_0269 gene and its upstream and downstream gene sequences in the genome sequence of A.ferrooxidans standard strain ATCC 23270 published in GenBank: CP001212.1:

[0174] HB1F:

[0175] 5′-CAC GAATTC CACGGACTGGGCGGATTAT-3′

[0176] HB1R:

[0177] 5′-CAC TCTAGA ATCACCCCTTGTCGGCAATG-3′

[0178] HB2F:

[0179] 5′-CCG TCTAGA GAGGAGTACTTAAGATGAGCAAACA-3′

[0180] HB2R:

[0181] 5′-GAT GAGCTC TGGTCGGTGATGACGGTACGG-3′

[0182] HB1F and HB1R primers, HB2F and HB2R primers wer...

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Abstract

The invention discloses an unmarked gene knock-out method of extremely acidophilic thiobacillus ferrooxidans based on the principle of homologous recombination. The method comprises the following steps of: constructing initial plasmids, suicide plasmids containing homologous fragments at upstream and downstream parts of target genes to be knocked out, and induction plasmids containing yeast endonuclease I-SceI genes; jointing and transferring the suicide plasmids and the induction plasmids to acidophilic thiobacillus ferrooxidans; and screening and identifying single commutators generating homologous recombination for the first time and double-exchange mutant strains generating homologous recombination for the second time. The method disclosed by the invention realizes the unmarked gene knock-out of acidophilic thiobacillus ferrooxidans for the first time, can realize the purpose of quickly, stably and efficiently knocking out the genes of the thiobacillus ferrooxidans, and can be used for researching the functional and metabolic mechanisms of the genes of the thiobacillus ferrooxidans, improving the genetic characters and constructing efficient bioleaching engineering bacteria; and moreover, the obtained mutant strain does not carry any resistance gene, thus the obtained mutant strain not only can be used as an original strain to knock out and improve genes in subsequent different sites, but also can be safely used for large-scale industrial production.

Description

technical field [0001] The invention relates to a gene knockout method based on the principle of homologous recombination, in particular to a marker-free gene knockout method for extreme acidophilic Thiobacillus ferrooxidans based on the principle of homologous recombination. Background technique [0002] Acidithiobacillus ferrooxidans (A.ferrooxidans for short) is a typical type of obligate chemoautotrophic bacteria, which can obtain ferrous ions (Fe 2+ ) and reducing sulfur compounds (RISCs) in the oxidation process to obtain energy for growth, is currently the dominant and efficient leaching bacteria species in the application of sulfide ore biometallurgy, and is also a model bacteria for studying the iron and sulfur oxidation metabolism mechanism of ore leaching microorganisms. However, A.ferrooxidans is obligately autotrophic, grows very slowly, has low cell yield, and is very difficult to genetically manipulate, which greatly limits the basic research and industrial ap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12R1/01
Inventor 刘相梅王慧妍于洋洋刘双双林建群林建强庞昕
Owner SHANDONG UNIV
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