CN-118726437-B - Acidophilic thiobacillus and gene traceless knockout method thereof

Abstract

The invention belongs to the field of microorganisms, and discloses an acidophilic thiobacillus and a gene traceless knockout method thereof, wherein the method comprises the following steps: recombining the I-SceI gene, the IPTG inducible promoter lacI-PtacO gene and the suicide plasmid pSDUDI, constructing a pSDUDI-LacI-I-SceI gene knockout plasmid, and expressing the I-SceI enzyme knockout I-SceI gene by IPTG induction; the method improves the probability of separating the gene knockout plasmid part from the chromosome of the acidophilic thiobacillus, simplifies the steps of operating genetic operation by a one-step method, consumes less time and shortens the gene knockout time.

Inventors

• CHEN LINXU
• GAO CHANG
• LIN JIANQUN

Assignees

• 山东大学

Dates

Publication Date

20260508

Application Date

20240731

Claims (9)

1. 1. A method for traceless knockout of an acidophilic thiobacillus gene, comprising: Recombining the I-SceI gene, the IPTG inducible promoter lacI-PtacO gene and the suicide plasmid pSDUDI to construct a pSDUDI-LacI-I-SceI gene knockout plasmid, and expressing the I-SceI enzyme knockout target gene by IPTG induction; Comprises double enzyme cutting suicide plasmid pSDUDI to obtain linearization fragment, connecting IPTG induced promoter lacI-P tacO gene fragment with linearization fragment by homologous recombinase to construct pSDUDI-LacI plasmid; connecting pSDUDI-LacI plasmid with I-SceI gene fragment by homologous recombinase to construct pSDUDI-LacI-I-SceI plasmid; connecting a homologous arm fragment of a target gene to be knocked out by the acidophilic thiobacillus with pSDUDI-LacI-I-SceI vector by using homologous recombinase to construct a gene knockout plasmid; The gene knockout plasmid is transformed into competent escherichia coli, then is jointed and transferred into acidophilic thiobacillus, and is screened to obtain the gene knockout acidophilic thiobacillus.
2. 2. The method of claim 1, wherein restriction enzymes MluI and NdeI are used to double-cleave suicide plasmid pSDUDI to obtain linearized fragment, or wherein homologous recombination enzyme is ExnaseII, or wherein IPTG inducible promoter-containing plasmid is used as template to obtain IPTG inducible promoter gene fragment lacI-P tacO by PCR amplification.
3. 3. The method of traceless knockout of a gene of thiobacillus acidophilus according to claim 1, wherein the thiobacillus acidophilus is a mesophilic thiobacillus acidophilus or a ferrous thiobacillus acidophilus.
4. 4. The method of traceless knockout of a gene of thiobacillus acidophilus according to claim 1, wherein suicide plasmid pSDUDI is fused with IPTG inducible promoter lacI-P tacO by homologous recombinase ExnaseII to construct pSDUDI-lacI plasmid, transformed and cultured.
5. 5. The method of traceless knockout of an acidophilic thiobacillus gene according to claim 4, wherein the reaction condition for fusing the suicide plasmid pSDUDI with the IPTG inducible promoter lacI-P tacO is 37 ℃ and the mixture is heated in a water bath for 30min.
6. 6. The method of traceless knockout of an acidophilic thiobacillus gene according to claim 1, wherein the plasmid pMSD1-I-SceI containing the endonuclease I-SceI gene fragment is used as a template for PCR amplification to obtain the I-SceI gene fragment; or, PCR amplifying the gene fragment of the homologous arm on the upstream and downstream of the gene to be knocked out by using the genome of the Acidithiobacillus as a template.
7. 7. The method of claim 1, wherein the linearized plasmid pSDUDI-LacI fragment and the I-SceI gene fragment are ligated using homologous recombinase ExnaseII to construct a pSDUDI-LacI-I-SceI plasmid, transformed, and cultured.
8. 8. The method of traceless knockout of a gene of Acidithiobacillus according to claim 1, wherein the target gene is pelD gene, the restriction enzymes SpeI and SacI double-cleave pSDUDI-LacI-I-SceI plasmid to obtain pSDUDI-LacI-I-SceI linearized plasmid, pSDUDI-LacI-SceI linearized plasmid is connected with upstream and downstream homology arm UHA and DHA gene fragments of pelD gene by homologous recombination enzyme ExnaseII to construct knockout plasmid pSDUDI-LacI-SceI-pelD.
9. 9. The method of traceless knockout of a gene of Acidithiobacillus according to claim 1, wherein the constructed knockout plasmid pSDUDI-LacI-I-SceI-pelD is transformed into competent cells of E.coli to obtain donor bacteria as a conjugation transfer, and the plasmid is transferred into recipient bacteria Acidithiobacillus according to the conjugation transfer method.

Description

Acidophilic thiobacillus and gene traceless knockout method thereof Technical Field The invention relates to the technical field of microorganisms, in particular to acidophilic thiobacillus and a gene traceless knockout method thereof. Background The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art. Acidophilic thiobacillus (acidophilus spp.) is a gram-negative autotrophic bacteria capable of obtaining energy by oxidizing elemental sulfur, reducing sulfide and ferrous iron, plays a very important role in bacterial leaching, can remarkably improve leaching efficiency, and makes the acidophilic thiobacillus become one of the strains with highest application value in the biological metallurgical industry. The acidophilic thiobacillus has the characteristics of harsh growth conditions, long generation time, slow growth and complex genetic operation, so that the research progress of the acidophilic thiobacillus is slower, and the acidophilic thiobacillus is mainly researched and modified by a joint transfer operation system. For the traditional genetic operation method of the acidophilic thiobacillus, related knockout research and reports are less, so the inventor establishes a traceless gene knockout method of the acidophilic thiobacillus caldarius based on the homologous recombination principle in the earlier research, see patent CN102604929A, which is a gene traceless knockout and integration method of the acidophilic thiobacillus caldarius, the knockout process is based on a conjugation transfer system, two plasmids are needed for at least two times of conjugation, and plasmid loss operation is needed, so that the knockout of the acidophilic thiobacillus caldarius gene can be realized, however, the inventor finds that the method still has the problems of complicated knockout process, complex genetic operation, long time consumption and the like, and is unfavorable for the research of the acidophilic thiobacillus. Disclosure of Invention Aiming at the defects existing in the prior art, the embodiment of the invention aims to provide the acidophilic thiobacillus and the gene traceless knockout method thereof so as to solve the problems of complex genetic operation, long time consumption and the like of the acidophilic thiobacillus. The first aspect of the invention discloses a traceless knockout method of an acidophilic thiobacillus gene, which comprises the following steps: The I-SceI gene, the IPTG inducible promoter lacI-PtacO gene and the suicide plasmid pSDUDI are recombined to construct a pSDUDI-LacI-I-SceI gene knockout plasmid, and the target gene is knocked out by IPTG induced expression of the I-SceI enzyme. The invention combines suicide plasmid containing endonuclease I-SceI enzyme cutting site and I-SceI enzyme expression plasmid, successfully constructs one-step knockout plasmid pSDUDI-LacI-I-SceI which can express I-SceI enzyme by IPTG induction, effectively shortens gene knockout time of the acidophilic thiobacillus, and provides a new approach for deep research and modification of the acidophilic thiobacillus. In some embodiments, suicide plasmid pSDUDI is subjected to double digestion to obtain a linearization fragment, and the IPTG-inducible promoter lacI-P tacO gene fragment is connected with the linearization fragment by using homologous recombinase to construct pSDUDI-LacI plasmid; connecting pSDUDI-LacI plasmid with I-SceI gene fragment by homologous recombinase to construct pSDUDI-LacI-I-SceI plasmid; connecting a homologous arm fragment of a target gene to be knocked out by the acidophilic thiobacillus with pSDUDI-LacI-I-SceI vector by using homologous recombinase to construct a gene knockout plasmid; The gene knockout plasmid is transformed into competent escherichia coli, then is jointed and transferred into acidophilic thiobacillus, and is screened to obtain the gene knockout acidophilic thiobacillus. In some embodiments, the suicide plasmid pSDUDI is double digested with the restriction enzymes MluI and NdeI to obtain a linearized fragment. In some embodiments, the homologous recombinase is EEnaseII. In some embodiments, the IPTG-inducible promoter gene fragment lacI-P tacO is obtained by PCR amplification using a plasmid containing the IPTG-inducible promoter as a template. In some embodiments, the thiobacillus acidophilus is a thiobacillus acidocaldarius (Acidithiobacillus caldus). In some embodiments, suicide plasmid pSDUDI is fused with IPTG-inducible promoter lacI-P tacO using homologous recombinase EEnaseII to construct pSDUDI-lacI plasmid, transformed, and cultured. In some embodiments, the suicide plasmid pSDUDI is fused to the IPTG-inducible promoter lacI-P tacO at 37 ℃ for 30min in a water bath. In some embodiments, the I-SceI gene fragment is obtained by PCR amplification using plasmid pMSD1-I-SecI containing the I-SceI gene fragment of the endonuclease as a template. In some embodiment