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Bacillus subtilis oscillating gene expression system, construction method and application thereof

A Bacillus subtilis, gene expression technology, applied in the biological field, can solve the problems of high cost of medium raw materials, restrictions on the application of streptococcus fermentation, low yield, etc.

Active Publication Date: 2019-08-06
汪洋
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Before the 1980s, hyaluronic acid was mainly extracted from cockscombs, the source of raw materials was limited, and the output was very low
The safety requirements of hyaluronic acid fermentation limit the fermentation application of Streptococcus, and Streptococcus has high nutritional requirements and the cost of medium raw materials is high

Method used

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  • Bacillus subtilis oscillating gene expression system, construction method and application thereof
  • Bacillus subtilis oscillating gene expression system, construction method and application thereof
  • Bacillus subtilis oscillating gene expression system, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1: Construction of recombinant Bacillus subtilis BS168ΔcomA knocking out the response regulator gene comA by double-crossover homologous recombination.

[0090] 1. the construction of bacillus subtilis recombinant plasmid pMAD-△comA comprises the following steps:

[0091] (1) Using the genomic DNA of Bacillus subtilis 168 (a commercially available conventional strain) as a template, primers comAupFor, comAupRev and comAdnFor, comAdnRev were used to amplify the upstream and downstream fragments of the comA gene, respectively.

[0092] (2) BamHI digestion of plasmid pMAD, recovery and purification.

[0093] (3) Perform Gibson assembly on the upstream and downstream homology arms of the comA gene purified and recovered in step (1) and the plasmid vector pMAD digested and linearized in step (2) to obtain the recombinant plasmid pMAD-△comA; the Gibson assembly system is comA 1.2 μl each of the upstream and downstream fragments of the gene, 0.6 μl of the linearized v...

Embodiment 2

[0117] Example 2: Transformation of the Bacillus subtilis mutant BS168ΔcomA with the oscillating gene expression regulatory plasmid pOSC to construct a Bacillus subtilis oscillating gene expression system.

[0118] 1. The construction of the oscillating gene expression regulation plasmid pOSC comprises the following steps:

[0119] (1) Using Bacillus subtilis 168 genomic DNA as a template, design primers PsrfAFor and PsrfARev to amplify the 0.6kb promoter sequence fragment upstream of the srfA gene, recover and purify.

[0120] (2) Using Escherichia coli DH10B (commercially available conventional strain) genomic DNA as a template, primers tetRFor and tetRRev were designed to amplify the tetR gene fragment, recovered and purified.

[0121] (3) NheI / XbaI double digestion plasmid pHT43, recovery and purification of 5.4Kb vector fragment;

[0122] (4) The promoter PsrfA fragment purified and recovered in step (1) and step (2), and the tetR gene fragment were connected to the plas...

Embodiment 3

[0135] Example 3: Application of Bacillus subtilis oscillatory gene expression system to express hyaluronic acid

[0136] 1. A method for constructing a regulatory plasmid expressing a hyaluronic acid oscillatory gene, specifically comprising:

[0137] (1) Using Streptococcus pyogenes (commercially available conventional strain) genomic DNA as a template, design primers hasAFor and hasARev to amplify hyaluronan synthase gene hasA, recover and purify.

[0138] (2) Using Bacillus subtilis genomic DNA as a template, design primers tuaDFor, tuaDRev, gtaBFor, gtaBRev to amplify UDP-glucose dehydrogenase gene tuaD and UTP-glucose-1-phosphate uridine acyltransferase gene gtaB respectively, recover and purify .

[0139] (3) BamH1 digestion of plasmid pOSC, recovery and purification.

[0140] 2. The hyaluronic acid synthase gene hasA, UDP-glucose dehydrogenase gene tuaD and UTP-glucose-1-phosphate uridine acyltransferase gene gtaB purified and recovered in step (1) and step (2) by Gi...

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Abstract

The invention discloses a bacillus subtilis oscillating gene expression system, a construction method and an application thereof, which belong to the technical field of biology. The bacillus subtilisoscillating gene expression system is obtained by knocking out a response regulatory factor gene comA on the Bacillus subtilis genome and transferring the gene into the oscillating gene expression regulatory plasmid pOSC. By regulating the cyclical switching process of gene expression, the oscillating gene expression system can alleviate the supply pressure of a metabolic substrate and energy during a stable growth period, the gene expression is balanced with the supply of the substrate, so that the expression yield us ubcreased. By using the system to express the hyaluronic acid synthesis-related gene, a recombinant Bacillus subtilis with high yield hyaluronic acid is successfully constructed.

Description

[0001] Technical field: [0002] The invention relates to a Bacillus subtilis oscillating gene expression system and its construction method and application, in particular to the construction process of a gene periodic switch regulated by comA / PsrfA and tetR / Ptet elements in Bacillus subtilis cells, belonging to the field of biotechnology . [0003] Background technique: [0004] With the development of molecular biology, microbiology and other disciplines, a variety of prokaryotic and eukaryotic expression systems have been developed to produce recombinant proteins. Among them, Bacillus subtilis, as an important Gram-positive model strain, has the characteristics of non-pathogenicity, clear genetic background, easy isolation and culture, and strong protein secretion ability. It is an ideal host for heterologous protein expression and secretion. So far, a large number of prokaryotic and eukaryotic genes have been cloned and expressed in Bacillus subtilis cells, some of which h...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N15/67C12N15/90C12N1/21C12P19/26C12R1/125
CPCC12N9/0006C12N9/1051C12N9/1241C12N15/67C12N15/75C12N15/902C12N2830/001C12P19/26C12Y101/01022C12Y204/01212C12Y207/07009
Inventor 汪洋丁艳东严强
Owner 汪洋
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