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Method for screening strains with high yield of arginine by using biosensor

A biosensor, arginine technology, applied in the biological field, can solve the problems of long detection cycle, time-consuming, labor-intensive, etc., and achieve the effects of short detection cycle, simple operation and high detection efficiency

Active Publication Date: 2019-12-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The existing method for screening high-yield arginine strains is mainly to carry out multiple rounds of random mutagenesis on arginine-producing strains, then culture the strains obtained by mutagenesis, and finally determine the amount of arginine produced by the Sakaguchi reagent method or high-performance liquid chromatography. The content of arginine in the fermentation supernatant obtained from the cultivation of a mutagenic strain, because the Sakaguchi reagent method or high performance liquid chromatography requires complex sample processing, and the detection cycle is relatively long, it is impossible to achieve the content of arginine in the fermentation broth. rapid detection, which makes the entire screening process labor-intensive and time-consuming

Method used

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  • Method for screening strains with high yield of arginine by using biosensor
  • Method for screening strains with high yield of arginine by using biosensor
  • Method for screening strains with high yield of arginine by using biosensor

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1: Construction of a biosensor that can be used to screen high-producing arginine strains

[0040] Specific steps are as follows:

[0041] Scheme 1: Using the genomic DNA of Corynebacterium crenatum SYPA5-5 as a template, argR-F1 (nucleotide sequence shown in SEQ ID NO. 4) and argR-R1 (nucleotide sequence shown in SEQ ID NO.5) is the primer, and the gene argR encoding the repressor protein is amplified by PCR (the nucleotide sequence is shown in SEQ ID NO.1); the amplified gene argR encoding the repressor protein is tapped and purified Then, it was ligated with the shuttle plasmid pDXW-10 that was digested with EcoRI and NotI to obtain the recombinant plasmid argR-pDXW-10. The recombinant plasmid argR-pDXW-10 was sequenced and verified, and the result showed that the ligation was successful.

[0042] Using the genomic DNA of Corynebacterium crenatum SYPA5-5 and the plasmid pK18mobsacB as templates, PargC-sacB-F1 (nucleotide sequence shown in SEQ ID NO.6) and PargC-sac...

Embodiment 2

[0043] Example 2: Preliminary verification of a biosensor that can be used to screen high arginine-producing strains

[0044] Specific steps are as follows:

[0045] The biosensor pSenArg-sacB and the biosensor pSenArg-gfp were electrotransformed into Corynebacterium crenatum SYPA5-5 to obtain recombinant Corynebacterium crenatum / pSenArg-sacB and recombinant Corynebacterium crenatum / pSenArg -gfp; Streak inoculate the recombinant Corynebacterium crenatum / pSenArg-sacB and the recombinant Corynebacterium crenatum / pSenArg-gfp on a solid plate without sucrose, culture for 48h at 30°C for activation to obtain a single colony ; With the recombinant Corynebacterium crenatum / pDXW-10 containing the empty plasmid of pDXW-10 as a positive control, pick a single colony of recombinant Corynebacterium crenatum / pSenArg-sacB, and streak it to contain 10g / L sucrose and respectively In the solid plates with arginine concentrations of 0, 10, 20, 40, 60, 80, and 100 mM, after culturing at 30°C for 48 ...

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Abstract

The invention discloses a method for screening strains with high yield of arginine by using a biosensor, and belongs to the technical field of biology. The invention provides the biosensor for screening the strains with high yield of the arginine, the biosensor comprises a gene argR encoding a repressor protein, a gene PargC encoding a promoter and a marker gene sacB, and the biosensor is used forscreening the strains with high yield of the arginine and has the advantages of simple operation, short detection period and high detection efficiency.

Description

Technical field [0001] The invention relates to a method for screening arginine high-producing strains by using a biosensor, and belongs to the field of biotechnology. Background technique [0002] Arginine is an essential amino acid for the human body and exists in many animal foods. Studies have shown that arginine is an important intermediate metabolite in the urea cycle of organisms, an important substance for the body to synthesize cytoplasmic proteins and nuclear proteins, or aspartic acid, glutamic acid, proline, hydroxyproline, Polyamines (putrescine, spermine), etc., are converted into high-energy phosphoric acid compounds, creatine phosphate intermediates, and have unique physiological and pharmacological effects. Therefore, they are widely used in food, medicine and cosmetics. [0003] At present, arginine is often produced by microbial fermentation in industry, and the reported arginine producing strains are mainly Corynebacterium crenatum, Corynebacterium glutamicum, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/31C12N15/65C12N1/21G01N30/02G01N21/33C12R1/15
CPCC07K14/34C12N15/63C12N15/65G01N21/33G01N30/02
Inventor 徐美娟陈佳敏饶志明刘平平林月阳江安杨套伟张显
Owner JIANGNAN UNIV