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A method for screening arginine high-yielding strains using a biosensor

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

Active Publication Date: 2021-03-30
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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  • A method for screening arginine high-yielding strains using a biosensor
  • A method for screening arginine high-yielding strains using a biosensor
  • A method for screening arginine high-yielding strains using a biosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0040] Specific steps are as follows:

[0041] Scheme one: with the genomic DNA of corynebacterium crenatum (Corynebacterium crenatum) SYPA5-5 as template, with argR-F1 (nucleotide sequence as shown in SEQ ID NO.4) and argR-R1 (nucleotide sequence as shown in SEQ ID NO.4) ID NO.5) is a primer, and the gene argR (nucleotide sequence shown in SEQ ID NO.1) encoding the repressor protein is obtained by PCR amplification; the amplified gene argR encoding the repressor protein is subjected to rubber tapping purification After that, it was ligated with the shuttle plasmid pDXW-10 that had been 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 results showed that the connection was successful.

[0042] Using the genomic DNA of Corynebacterium crenatum SYPA5-5 and the plasmid pK18mo...

Embodiment 2

[0043] Example 2: Preliminary verification of biosensors 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 respectively to obtain recombinant Corynebacterium crenatum / pSenArg-sacB and recombinant Corynebacterium crenatum / pSenArg -gfp; Streak inoculation of recombinant Corynebacterium crenatum / pSenArg-sacB and recombinant Corynebacterium crenatum / pSenArg-gfp on a solid plate without sucrose, culture at 30°C for 48 hours for activation, and obtain a single colony ; With the recombinant Corynebacterium crenatum / pDXW-10 containing the pDXW-10 empty plasmid as a positive control, pick a single colony of the recombinant Corynebacterium crenatum / pSenArg-sacB, streak to contain 10g / L sucrose and contain respectively In the solid plate with arginine concentration of 0, 10, 20, 40, 60, 80, 100mM, after culturing...

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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 high-arginine-producing strains by using a biosensor, and belongs to the field of biotechnology. Background technique [0002] Arginine is an essential amino acid found in many animal foods. Studies have shown that arginine is an important intermediate metabolite of the urea cycle in organisms, an important substance for the body to synthesize cytoplasmic proteins and nuclear proteins, or aspartic acid, glutamic acid, proline, hydroxyproline, Polyamines (putrescine, spermine) and other intermediates converted into creatine phosphate, a high-energy phosphate compound, have unique physiological and pharmacological effects. Therefore, they are widely used in the fields of food, medicine and cosmetics. [0003] At present, arginine is often produced industrially through microbial fermentation, and the reported arginine-producing strains mainly include Corynebacterium obstatus, Corynebacterium glutamicum, Esc...

Claims

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Application Information

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Patent Type & Authority Patents(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
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