Dehydrated tetracycline-induced escherichia coli-bacillus subtilis universal inducible expression system

An expression system and expression cassette technology, applied in the field of genetic engineering, can solve problems such as the inability to construct a general induction and regulation system of large intestine-subtilis subtilis, inability to induce constitutive expression, gene sequence mutation, etc., and achieve simple structure, strict regulation and high activity Effect

Active Publication Date: 2022-06-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although induction systems such as IPTG and xylose are widely used in Bacillus subtilis, due to the disadvantages that xylose is used as a carbon source, IPTG is toxic to the human body, the expression intensity is limited, and constitutive expression cannot be induced in E. coli. There are certain problems in actual use; for example, when expressing some toxic proteins, the leaky expression in E. coli often causes mutations in the gene sequence and premature termination of expression, and it is impossible to construct a general induction and regulation system of E. coli-subtilis

Method used

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  • Dehydrated tetracycline-induced escherichia coli-bacillus subtilis universal inducible expression system
  • Dehydrated tetracycline-induced escherichia coli-bacillus subtilis universal inducible expression system
  • Dehydrated tetracycline-induced escherichia coli-bacillus subtilis universal inducible expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Induced expression plasmid pHT-P 21 -tet-RBS-P tet - Construction of sfGFP and engineering strains

[0044] like figure 2 As shown, the constructed inducible expression plasmid pHT-P 21 -tet-RBS-P tet -sfGFP mainly consists of three parts: (1) commercial plasmid pHT01 expression vector; (2) repressor protein tetR expression cassette; (3) superfolded green fluorescent protein (sfGFP) expression cassette.

[0045] Specific steps are as follows:

[0046] (1) Construction of pHT plasmid backbone

[0047] Using primers pHT-LiF: 5'-ctgcaggtcgacgtcccc-3' and pHT-LiR: 5'-agcgcaacgcaattaatgtga-3', the backbone of the pHT plasmid was amplified using the commercial plasmid pHT01 as a template.

[0048] (2) Construction of repressor protein tetR expression cassette

[0049] Chemically synthesized repressor protein tetR expression cassette ( figure 1 ); The repressor protein tetR expression frame is made of subtilis constitutive promoter P21, RBS sequence and rep...

Embodiment 2

[0060] Example 2: Universal inducible expression plasmid pHT-P 21 -F119Y-RBS0-P tet - Construction of sfGFP and engineering strains

[0061] Specific steps are as follows:

[0062] (1) Preparation of mutant plasmid pHT-P 21 -F119Y-RBS0-P tet -sfGFP preparation

[0063] With the plasmid pHT-P that embodiment 1 prepares 21 -tet-RBS-P tet -sfGFP is used as a template, the phenylalanine at position 119 of the repressor protein tetR on the plasmid is mutated into tyrosine (F119Y), and the RBS in the expression frame of the repressor protein tetR on the plasmid is changed to RBS0 at the same time (the nucleotide sequence is as follows shown in SEQ ID NO.9).

[0064] The primer sequences involved are as follows:

[0065] use primers

[0066] RBS0-F: 5'-attcctccttattattatgtagttactatataaaagcattagtgtatcaattccacga-3';

[0067] RBS0-R: 5'-ctacataaataaggaggaatcacatgtccagattagataaaagtaaagtgattaac-3'.

[0068] F119Y-F: 5'-agtgaaaaaccttgttggcataaa t aggctaattgattttcgagagttt-3',

...

Embodiment 3

[0074] Example 3: Induced expression plasmid pHT-P 21 -tet-RBS0-P tet - Construction of sfGFP and engineering strains

[0075] Specific steps are as follows:

[0076] (1) pHT-P 21 -tet-RBS0-P tet - Construction of sfGFP

[0077] With the recombinant plasmid pHT-P that embodiment 1 prepares 21 -tet-RBS-P tet -sfGFP is used as a template, and only the RBS in the repressor protein tetR expression frame on the plasmid is changed to RBS0 (the nucleotide sequence is shown in SEQ ID NO.9), and the primer sequence used is as follows: RBS0-F: 5'-attcctccttattattgtagttactatataaaagcattagtgtatcaattccacga- 3';

[0078] RBS0-R: 5'-ctacataaataaggaggaatcacatgtccagattagataaaagtaaagtgattaac-3'.

[0079] Carry out the fragment that PCR (reaction condition is the same as embodiment 2) obtains construct recombinant plasmid pHT-P by Seamless Cloning Kit (Beyotime Biotechnology) 21 -tet-RBS0-P tet -sfGFP, verified by sequencing, confirmed that the recombinant plasmid was successfully const...

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Abstract

The invention discloses a dehydration tetracycline induced escherichia coli-bacillus subtilis universal type inducible expression system, and belongs to the field of gene engineering. According to the invention, an operon Pet expression system capable of using anhydrotetracycline to induce expression in escherichia coli and bacillus subtilis is constructed, and the system takes a commercial plasmid pHT01 as an expression vector and comprises a repressor protein tetR expression cassette and a super-folded green fluorescent protein (sfGFP) expression cassette. Wherein the gene sequence and the RBS sequence of the repressor protein tetR in the expression cassette of the repressor protein tetR are mutated to change the affinity and the expression intensity of the repressor protein. The operon Pet expression system shows low leakage and high induction magnification times in escherichia coli and bacillus subtilis. Therefore, the general expression system is simple in structure, high in activity and rigorous in regulation and control, and has a wide application prospect in heterologous protein high-efficiency expression and synthetic biology research.

Description

technical field [0001] The invention relates to an anhydrotetracycline-induced Escherichia coli-Bacillus subtilis universal inducible expression system, which belongs to the field of genetic engineering. Background technique [0002] As a class of Gram-positive bacteria, Bacillus subtilis is widely used in the production of various protease preparations and valuable proteins due to its good biological safety, strong secretion ability, and no codon preference. There are a variety of expression systems for expressing heterologous proteins in Bacillus subtilis, which are divided into different promoter characteristics, mainly including self-inducible expression systems, constitutive expression systems, and inducible expression systems. Promoters are mainly divided into two types: constitutive promoters and inducible promoters. Constitutive promoters can express foreign genes at all stages of bacterial growth, while inducible promoters show no activity or low activity when they...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/75C07K14/245C12N15/31C07K14/435C12R1/19C12R1/125
CPCC12N15/70C12N15/75C07K14/245C07K14/43595C12N2800/60Y02A50/30
Inventor 刘龙陈坚吕雪芹堵国成李江华刘延峰李洋武耀康
Owner JIANGNAN UNIV
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