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Method for screening unmarked gene knockout bacterial strain of acidithiobacillus thiooxidans

A Thiobacillus thiooxidans, marker-free gene technology, applied in biochemical equipment and methods, microbial determination/inspection, hybrid cell preparation, etc., can solve the problems of low screening efficiency and large workload.

Active Publication Date: 2013-08-07
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the lack of marker-free gene knockout technology for acidophilic Thiobacillus thiooxidans and the existing marker-free gene knockout technology for acidophilic Thiobacillus ferrooxidans and acidophilus thermophilic Thiobacillus, there are low screening efficiency and heavy workload problem, the present invention provides a high-efficiency screening method for marker-free gene knockout strains of acidophilic thiothiooxidans (A.thiooxidans)

Method used

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  • Method for screening unmarked gene knockout bacterial strain of acidithiobacillus thiooxidans
  • Method for screening unmarked gene knockout bacterial strain of acidithiobacillus thiooxidans
  • Method for screening unmarked gene knockout bacterial strain of acidithiobacillus thiooxidans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Marker-free knockout of the multicopper oxidase gene (cueO) of the extreme acidophilic Thiobacillus thiooxidans

[0045] 1. Construction of knockout plasmid backbone pZK19

[0046] (1) Construction of plasmid pK19

[0047] According to the sequence information of plasmid pUC19, primers were designed to amplify the oriColE1 part of its replication origin:

[0048] 19F: 5′-CAGC GAGCTC GGGATAACGCAGGAAAGA-3′

[0049] 19R: 5′-CAAA GGGCCC TAATAGACTGGATGGAGGCG-3′

[0050] A Sac I restriction site was added to the 5' end of primer 19F, and an Apa I restriction site was added to the 5' end of primer 19R. The two primers use the plasmid pUC19 as a template to amplify the fragment with pUC19oriColE1 by PCR (polymerase chain reaction). The composition of the PCR reaction system (25uL) is as follows: 5×PrimerSTAR Buffer (Mg 2+ plus) 5uL; dNTP Mixture (2.5mM each) 2uL; primer 19F (10uM) 0.25uL; primer 19R (10uM) 0.25uL; plasmid pUC19 template 0.25uL; PrimerSTAR H...

Embodiment 2

[0112] Example 2: Marker-free knockout of the MerR family regulatory protein gene (cueR) of Acidophilus thiothiooxidans

[0113] 1. Construction of A.thiooxidans cueR Gene Knockout Plasmid

[0114] A.thiooxidans cueR gene knockout flowchart see Figure 6 . The construction of the knockout plasmid backbone pZK19 is the same as in Example 1 (omitted).

[0115] According to the genome sequence information of A.thiooxidans standard strain ATCC19377 published on NCBI, primers were designed to amplify the upper and lower parts of the cueR gene respectively as the upper and lower homology arms of the knockout gene:

[0116] RUHF: 5′-AA GTC GAC CGTGCGTGATGTGGGTTAT-3′

[0117] RUHR: 5′-CC AAGCTT GACTGAATTTTCACGCTCC-3′

[0118] RDHF: 5′-CC AAGCTT TGGGATGTGAGTCGGGATC-3′

[0119] RDHR: 5′-TT TCTAGA GGGTCACCAGCGCGGGAAC-3′

[0120] A Sal I restriction site was added to the 5' end of the primer RUHF, a Hind III restriction site was added to the 5' end of the primers RUHR and R...

Embodiment 3

[0141] Example 3: Unmarked Knockout of Acidophilus Thiobacillus thiooxidans copA Gene

[0142] 1. Construction of A.thiooxidans copA Gene Knockout Plasmid

[0143] A.thiooxidans copA gene knockout flow chart see Figure 7 . The construction of the knockout plasmid backbone pZK19 is shown in Example 1 (omitted).

[0144] According to the genome sequence information of A.thiooxidans standard strain ATCC19377 published on NCBI, primers were designed to amplify the upper and lower parts of the copA gene respectively as the upper and lower homology arms of the knockout gene:

[0145] AUHF: 5′-TAT GTC GAC CGATCCAGTGCCATTTCCAG-3′

[0146]AUHR: 5′-GTG AAGCTT CTGCAGGAAGCACAGGTCATC-3′

[0147] ADHF: 5′-CTC AAGCTT CGTTTGAAGTGGCTGCGTC-3′

[0148] ADHR: 5′-CTT TCTAGA AGCCAGGTTGCCAGACCATC-3′

[0149] A Sal I restriction site was added to the 5' end of the primer AUHF, a Hind III restriction site was added to the 5' end of the primers AUHR and ADHF, and an Xba I restriction si...

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Abstract

The invention discloses a method for screening an unmarked gene knockout bacterial strain of acidithiobacillus thiooxidans. The method comprises introducing a knockout plasmid for construction of a target gene and an induction plasmid carrying an I-SceI gene into acidithiobacillus thiooxidans by conjugational transfer, carrying out single recon screening by a resistance maker, and carrying out double recon preliminary screening by blue-white selection. The method discloses the method for unmarked gene knockout of acidithiobacillus thiooxidans first, utilizes beta-galactosidase blue-white selection for double recon screening, can fast, stably and efficiently knock out a target gene, solves the problem that in the existing unmarked gene knockout process, selective pressure lacks so that screening is difficult, shortens double recon screening time, improves screening efficiency, reduces a screening cost and provides a novel method for stable genetic modification of acidithiobacillus thiooxidans.

Description

technical field [0001] The invention relates to a marker-free gene knockout method based on the principle of homologous recombination and a double-exchanger colony screening method based on β-galactosidase activity, in particular to an efficient screening method for acidophilic Thiobacillus thiooxidans (A.thiooxidans ) method for marker-free knockout strains. Background technique [0002] Acidithiobacillus thiooxidans (A.thiooxidans for short) is an extremely acidophilic, Gram-negative, obligate chemoautotrophic bacterium that can utilize various reducing or partially reducing inorganic sulfides (RISCs) ) to obtain the energy required for growth and reproduction, and to fix CO in the air through the Calvin cycle 2 as the main carbon source. It has played an important role in bacterial metallurgy and coal desulfurization. As an important leaching microorganism, combined with acidophilic Thiobacillus ferrooxidans (A.ferrooxidans) can significantly improve the latter's leachi...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12Q1/68C12N15/09C12N15/03
Inventor 刘相梅文晴王慧妍林建群
Owner SHANDONG UNIV
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