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N-acetylneuraminic acid specific biosensor and application thereof

An acetylneuraminic acid and biosensor technology, which is applied in the field of genetic engineering, can solve the problem of lack of key genes of N-acetylneuraminic acid, etc., and achieves the effects of simple construction method, good application prospect and easy use.

Active Publication Date: 2019-08-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ammonia glucose metabolism pathway in Bacillus subtilis is tightly regulated, does not form the accumulation of ammonia sugar, and there is no key gene for N-acetylneuraminic acid

Method used

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  • N-acetylneuraminic acid specific biosensor and application thereof

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Effect test

Embodiment 1

[0033] The construction of embodiment 1 recombinant plasmid

[0034]According to the plasmid p43NMK-AGE-NeuB (Zhang XL, Liu YF, Liu L, Wang M, Li JH, Du GC, ChenJ. Modular pathway engineering of key carbon-precursor supply-pathways for improved N-acetylneuraminic acid production in Bacillus subtilis. Biotechnology and bioengineering.2018; 115(9):2217-2231) sequence information, designed primers AGE-NeuB-F: 5'-CCGGGATCCATGGGCAAAAACTTACAAGCTCTGGCCCAGCTTTATAAAAATGCCC-3', AGE-NeuB-R: 5'-TCCCTGGTTTTTAAATTCGCTATGGATGATAAGTTCATCCGGGCAAGTCTT' Plasmid p43NMK-AGE-NeuB is used as a template to amplify N-acetylglucosamine isomerase (AGE) shown in SEQ ID NO.5 and N-acetylneuraminic acid synthase (NeuB) shown in SEQ ID NO.6 ; According to the plasmid pSTOP1622 sequence (SEQ ID NO.8), design vector reverse amplification primers: pSTOP1622-F:5'-GCGAATTTAAAAACCAGGGAGAATAAGGTACCGGCCGCATGCCG-3', pSTOP1622-R:5'-TTTTTGCCCATGGATCCCGGGAGCTCCGGAGATCTTCGAACTAGTTTGGACC-3', use the above primers to plas...

Embodiment 2

[0035] Embodiment 2 Construction of recombinant Bacillus subtilis strain B6S

[0036] The constructed recombinant plasmid pSTOP16220AGE-NeuB was transformed into Bacillus subtilis (Bacillus subtilis 168ΔnagPΔnagPΔgamPΔgamAΔnagAΔnagBΔ1dhΔpta::lox72; Δctc::Gna1). AGE-NeuB-F and AGE-NeuB-R were used for colony PCR, and a 2233bp band appeared, verifying that the recombinant Bacillus subtilis B6S was successfully constructed.

Embodiment 3

[0037] Example 3 Construction of Biosensor Plasmids

[0038] According to the sequence information of E. coli (Escherichia coli K12), design primers NanR-F: 5'-CGCAGGCGTTTTGTAATAAATTATTTCTTTTTGTTGGTGGTCTGACCGAAAGCGT-3', NanR-R: 5'-TGGGATTGATAGAATATGACATGGGCCTTATGAACGC-3', use the above primers to amplify NanR with E. coli K12 as a template Gene; according to the sequence of Bacillus subtilis (Bacillus subtilis) 168, primers were designed rapH-F: 5'-GTCATATTCTATCAATCCCAAATTTTGAAAACAATATAGAAGGAAG-3', rapH-R: 5'-TCATCCTATTCCTTGATCCTGTTTAAGGGG-3', phbs-F: 5'-AGGATCAAGGAATAGGATGAAAAAAGGAAAAAAAGGAATATTCGTTCG , phbs-R: 5'-AGTTCTTCTCCCTTACCCATCCCAAAAGGATACATTCAGTTCGTTTTATTATCATTTCTTCACAGTTCAG-3', using Bacillus subtilis (Bacillus subtilis) 168 as a template to amplify the above promoter; design primers according to the GFP gene sequence, GFP-F: 5'-ATGGGTAAGGGAGAAGAACTTTTCACTGGAGTTGT-3', GFP -R:5'-CCGGCAGTCTGACAAGTTATTTATTTGTATAGTTCATCCATGCCATGTGTAATCCCAGC-3',使用GFP基因为模板扩增后,根据pHT01质粒序列设...

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Abstract

The invention discloses an N-acetylneuraminic acid specific biosensor and application thereof and belongs to the field of genetic engineering. The N-acetylneuraminic acid specific biosensor is constructed with transcriptional regulation factor NanR and GFP (green fluorescent protein) gene, from Escherichia coli K12, acting as reporter genes; an effective relationship between N-acetylneuraminic acid and fluorescence intensity is established. The N-acetylneuraminic acid specific biosensor has an important application prospect in screening microorganisms which produce target metabolites.

Description

technical field [0001] The invention relates to a specific biosensor of N-acetylneuraminic acid and its application, belonging to the field of genetic engineering. Background technique [0002] N-acetylneuraminic acid is a kind of monosaccharide in organisms, which widely exists in microorganisms and mammals. In the human body, N-acetylneuraminic acid is the first contact site for cell information transmission, and participates in multiple physiological processes such as cell recognition and signal transduction. Therefore, N-acetylneuraminic acid is widely used to regulate IgG anti-inflammatory activity, enhance infant immunity, and promote infant brain development. At present, N-acetylneuraminic acid is mainly extracted from relatively rich natural materials such as casein and bird’s nest. The products obtained are likely to cause allergic reactions, or they are synthesized by chemical methods under harsh conditions, and the high-temperature and high-pressure processes are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/65C12N1/21C12R1/125
CPCC12N15/65C12N15/75C12N2830/002
Inventor 刘延峰堵国成李江华钱蕾张忠杰袁莉莉陈坚
Owner JIANGNAN UNIV
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