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A recombinant plasmid, its construction method and its application to precise genome transformation of mycobacteria

A construction method and plasmid technology, which is applied in plant genetic improvement, genetic engineering, recombinant DNA technology, etc., can solve problems such as low efficiency and difficulty in obtaining target DNA strains

Active Publication Date: 2020-12-11
南通汇成生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The efficiency of this method is usually low in actual use, and it is difficult to obtain the strains modified by the target DNA

Method used

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  • A recombinant plasmid, its construction method and its application to precise genome transformation of mycobacteria
  • A recombinant plasmid, its construction method and its application to precise genome transformation of mycobacteria
  • A recombinant plasmid, its construction method and its application to precise genome transformation of mycobacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 Construction of basic plasmids for precise genome editing based on mycobacteria

[0087] like figure 1 The method process shown is as follows:

[0088] Step 1, use BglII and NheI to double digest the pEN4.1A-T10M plasmid (the pEN4.1A-T10M plasmid is the prior art and can be obtained through procurement) to obtain the Pimyc-tetRDNA fragment.

[0089] Use BamHI and XbaI to double digest the pBlueScript SK(-) plasmid (pBlueScript SK(-) plasmid is an existing technology and can be obtained through purchase), and add the Pimyc-tetRDNA fragment at the same time to obtain pKS-Pimyc-tetR .

[0090] Step 2, based on the I-sceI gene, on the basis of a comprehensive analysis of the codon usage of mycobacteria such as Mycobacterium tuberculosis, Mycobacterium smegmatis and Mycobacterium fortuitum, I-sceI was optimized and synthesized using the gene designer software. The sceI gene is named sceM (702bp, SEQ ID NO: 1). Use NdeI and KpnI to double digest the pLU101 p...

Embodiment 2

[0102] This embodiment is an alternative technical solution of Embodiment 1. The distinguishing technical measures are only:

[0103] Using the chromosomal DNA of Mycobacterium denticola as a template, using primers

[0104] psmyc_F:tttaAGATCTGGATCGTCGGCACCGTCA and

[0105] psmyc_R: tttaGGTACCGGAT CGTGCTCATTTCGGGC, about 300 bp Psmyc promoter fragment was obtained by PCR, and cloned into pEN4.1A-T10M plasmid with BglⅡ and kpnⅠ restriction enzymes to replace the Pimyc promoter.

[0106] Because the tetracycline-inducible promoter has the problem of background expression and induction efficiency, the two need a certain balance. The main purpose is to adjust the background expression efficiency of the sceM gene, but the above two promoters can be used normally after replacement.

Embodiment 3

[0108] Example 3 Construction Targeting plasmid pMK5228 for precise knockout of MSMEG_5228

[0109] like image 3 As shown, the pMK5228 plasmid construction flow chart, the construction process is as follows:

[0110] (1) using (primer

[0111] SC_U_F:tttagaattcAACCCGGTGTGCGATCTGGT,

[0112] SC_U_R:ATCGCAGATGTCGCCCGTG;

[0113] SC_D_R1:ACCCTGTTATCCCTAGGGTTCTGCGAGGACGACA,

[0114] SC_D_R: GGGTTCTGCG AGGACGACA,

[0115] SC_D_R3: atttTCTAGAGCGTTAATTAAACTAGTAGATCTAAGCTTGATTACCCTGTTATCCCTAGGGTTCTGCG AGGACGACA)

[0116] PCR integration obtained knockout recombination exchange arms with upstream and downstream of MSMEG_5228 of 820bp and 946bp respectively, after HindⅢ and EcoRI digestion, pMK101 was double-digested with HindⅢ and EcoRI ( Obtained from the construction method of Example 1 ) connection to obtain the pMK1011 plasmid. The apramycin resistance gene of pIJ773 plasmid was digested with Xba I, and cloned into pMK1011 plasmid to obtain plasmid pMK1012. The pGOAL19 ...

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Abstract

The invention relates to a genome transformation technology and discloses a recombinant plasmid, a construction method and accurate genome transformation for mycobacterium. The technical scheme relates to plasmid construction capable of carrying out accurate genome editing in mycobacterium and an application thereof in continuously accurate gene knockout in the mycobacterium. By using the method, the length of a target DNA fragment for genome editing of mycobacterium can be at the bp-Mb level in theory, and no scar is left on a genome after transformation. Through construction of the method, vitally important genome editing means can be provided for the research on pathogenesis of high pathogenic mycobacterium and genome transformation of an industrial strain of important mycobacterium for producing an important steroid hormone prodrug.

Description

technical field [0001] The present invention relates to genome modification technology. Background technique [0002] Since the 1980s, phytosterol microbial transformation production precursors such as 4AD, 9-OH-AD and ADD have gradually become the raw materials for the synthesis of most steroid drugs (androgens, steroids, estrogens and corticosteroids). Mycobacterium has a complex cholesterol metabolism pathway, which can metabolize plant-derived sterol side chains with complex components. After decades of mutation breeding and genetic engineering, it has gradually become a major industry for the efficient and economical production of such intermediates strain. [0003] Tuberculosis is a serious public health problem that has plagued the world for many years. The disease is caused by Mycobacterium tuberculosis and kills about 2 million people every year. Mycobacterium tuberculosis grows slowly and can grow on cholesterol as the only carbon source. It is difficult to study...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N15/70C12N15/66C12N15/65
CPCC12N9/22C12N15/65C12N15/66C12N15/70
Inventor 路志群邢述永杜艳杨晓章冯永华
Owner 南通汇成生物科技有限公司
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