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Recombinant plasmid, construction method and accurate genome transformation for mycobacterium

A technology of mycobacteria and recombinant plasmids, which is applied in the direction of plant genetic improvement, genetic engineering, recombinant DNA technology, etc., and can solve the problems of difficulty in obtaining target DNA strains and low efficiency

Active Publication Date: 2017-10-10
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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  • Recombinant plasmid, construction method and accurate genome transformation for mycobacterium
  • Recombinant plasmid, construction method and accurate genome transformation for mycobacterium
  • Recombinant plasmid, construction method and accurate genome transformation for mycobacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0088] Step 1. Use BglII and NheI to double digest pEN4.1A-T10M plasmid (pEN4.1A-T10M plasmid is an existing technology and can be obtained through purchase) to obtain Pimyc-tetRDNA fragment.

[0089] The pBlueScript SK(-) plasmid was digested with BamHI and XbaI (the pBlueScript SK(-) plasmid is an existing technology and can be obtained through purchase), and the Pimyc-tetRDNA fragment was added and ligated pKS-Pimyc-tetR .

[0090] Step 2. Based on the I-sceI gene, on the basis of comprehensive analysis of the codon usage of Mycobacterium tuberculosis, Mycobacterium smegmatis and Mycobacterium fortuitum, the gene designer software is used to optimize the synthesis of I- The scel gene was named sceM (702bp, SEQ ID NO: 1). Use NdeI and KpnI double enzyme digestion of pLU101 plasmid (it is the prior art, disclosed in:...

Embodiment 2

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

[0103] Using chromosomal DNA of Mycobacterium tartaricum as template, using primers

[0104] psmyc_F: tttaAGATCTGGATCGTCGGCACCGTCA and

[0105] psmyc_R: tttaGGTACCGGAT CGTGCTCATTTCGGGC, a Psmyc promoter fragment of about 300 bp was obtained by PCR, which was digested and cloned into pEN4.1A-T10M plasmid using BglII and kpnI, which can replace Pimyc promoter.

[0106] Because the tetracycline-induced promoter has the problem of background expression and induction efficiency, the two need to be balanced. The main purpose is to regulate 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 Accurately knock out the targeting plasmid pMK5228 of MSMEG_5228

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

[0110] (1) Use (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 to obtain knockout recombination exchange arms of 820bp and 946bp upstream and downstream of MSMEG_5228 respectively, HindⅢ and EcoRI digestion with HindⅢ and EcoRI double digestion pMK101( Obtained from the construction method of Example 1 ) Ligation to obtain pMK1011 plasmid. The apramycin resistance gene of pIJ773 plasmid was obtained by digestion with XbaI, and cloned into pMK1011 plasmid to obtain plasmid pMK1012. PacI digests pGOAL19 plasmid (pG...

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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, the microbial transformation and production of plant sterol precursors such as 4AD, 9-OH-AD and ADD have gradually become the synthetic raw materials for most steroid drugs (androgens, steroids, estrogen and corticosteroids). Mycobacterium has a complex cholesterol metabolism pathway, which can metabolize plant-derived sterol side chains with complex components. After decades of mutagenesis and genetic engineering, it has gradually become the main industry for efficiently and economically producing such intermediates. Strains. [0003] Tuberculosis is a serious public health problem that has plagued the world for many years. The disease is caused by Mycobacterium tuberculosis and causes about 2 million deaths every year. Mycobacterium tuberculosis grows slowly and can grow with cholesterol as the only carbon source. It is very difficult to study i...

Claims

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Application Information

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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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