Unmarked gene knock-out method of extremely 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 problems of less antibiotic availability, antibiotic resistance gene residues, and inconvenient multi-gene knockout Research and other issues
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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: Marker-free knockout of the metallo-beta-lactamase gene (AFE 0269 gene, abbreviated as sdo) of Thiobacillus ferrooxidans extreme acidophilus
[0170] (1) Construction of suicide plasmid for knocking out A. ferrooxidans ATCC 23270 AFE_0269 gene
[0171] 1. Construction of the initial plasmid for A. ferrooxidans gene knockout (same as Example 1, omitted)
[0172] 2. Construction of suicide plasmid for knocking out AFE_0269 gene
[0173] Primers were designed 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 by 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] The HB1F and HB1R primers, HB2F and HB2R primers, re...
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