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Theophylline induction type gene expression system

A technology of gene expression and expression vector, applied in the field of theophylline-inducible gene expression system, can solve problems such as difficulty in large-scale preparation, increase in complexity of recombination system, etc.

Inactive Publication Date: 2017-04-26
JIANGNAN UNIV
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Problems solved by technology

Increased complexity of using these systems to construct recombinant systems in Bacillus subtilis
Moreover, the gene-induced expression system using the above-mentioned promoter elements needs to add the required chemical agents during the culture process to activate gene transcription and start protein translation, so it is not easy to achieve large-scale production

Method used

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  • Theophylline induction type gene expression system
  • Theophylline induction type gene expression system
  • Theophylline induction type gene expression system

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Embodiment 1

[0013] (1) Construction of B.subtilis / pBSG11 and B.subtilis / p43E-gus recombinant bacteria

[0014] The P43-riboE1 gene with the optimized sequence shown in SEQ ID NO.1 was subjected to whole gene synthesis, digested with BamHI / SalI, and cloned into pUC19 plasmid to obtain pUC57A plasmid. Using pUC57A as a template, the P43-riboE1 sequence was amplified, recovered as a large primer, inserted into the srfA promoter position of the pBSG03 plasmid, and replaced the srfA promoter to construct a pBSG11 recombinant plasmid. The pBSG11 recombinant plasmid transformed B. subtilis 168 to obtain the recombinant strain B. subtilis / pBSG11. For the construction method of pBSG03, please refer to Chengran Guan, Wenjing Cui*, Jiantao Cheng, LiZhou, Junling Guo, Xu Hu, Guoping Xiao, Zhemin Zhou*. Construction and development of an auto-regulatory gene expression system in Bacillus subtilis. Microb CellFact.2015, 14(1):150.

[0015] With primer gus F-homo:

[0016] CAAAACCCCCCTTTGCTGAGGTGGCAG...

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Abstract

The invention discloses a theophylline induction-type gene expression system, which belongs to the technical field of microbial biomass. The system takes a composition-type strong promoter P43 as a basic component, merges a theophylline controlling element riboE1 with reconstructed interval zone length among an artificial SD element and a SD sequence and an initial code, constructs the theophylline induction-type gene expression element P43-riboE1, and clones a sequence to a bacillus subtilis expression vector pBSG, the induction expression carrier pBSG11 is constructed, host 168 bacterium is conversed, and high-level and high inductivity expression of exogenous gene under theophylline control is realized. The high-efficiency controllable recombination expression of report gene GFP, GUS and aspartase is realized by using the recombinant bacteria. Compared with inductive agents such as xylose and IPTG, the system has the advantages of no toxicity, and low cost, and has wide application space by taking theophylline as the gene expression system of the inductive agent.

Description

technical field [0001] The invention relates to a theophylline-inducible gene expression system, which belongs to the field of microbial biotechnology. Background technique [0002] Inducible gene expression has important applications in both prokaryotic and eukaryotic heterologous gene expression systems. The commonly used inducible gene expression elements in Bacillus subtilis are the hybrid Pgrac promoter induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and the PxylA promoter induced by xylose . Although these gene expression elements can realize the expression of exogenous genes in Bacillus subtilis, affected by the gene regulation mode, these promoters need to introduce an exogenous repressor protein at the same time to realize the rest-induced transition. The complexity of using these systems to construct recombinant systems in Bacillus subtilis has increased. Moreover, the gene-induced expression system using the above-mentioned promoter elements needs to add ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/75C12N1/21C12R1/125
CPCC07K14/32C12N15/75C12N2800/101C12N2830/002C12N2840/002
Inventor 周哲敏崔文璟程锦涛韩来闯周丽刘中美索菲娅
Owner JIANGNAN UNIV
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