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Homoserine biosensor as well as construction method and application thereof

A biosensor, homoserine technology, applied in the biological field, can solve the problems of lack of detection methods, restrictions on high-throughput screening and breeding of L-homoserine high-yielding strains, etc.

Active Publication Date: 2021-02-19
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there is no report on the L-homoserine biosensor
Due to the lack of simple and fast detection methods, the high-throughput screening and breeding of L-homoserine high-yielding strains are greatly limited

Method used

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  • Homoserine biosensor as well as construction method and application thereof
  • Homoserine biosensor as well as construction method and application thereof
  • Homoserine biosensor as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: Construction of L-homoserine biosensor plasmid

[0023] In this example, each component module of the L-homoserine biosensor was amplified by PCR to obtain nucleotide fragments, and the fragments were connected in a certain order by molecular cloning technology to complete the construction of the L-homoserine biosensor plasmid. Wherein, the biosensor is composed of the transcriptional regulator NCgl0581 coding gene (Genebank: NP_599842) derived from Corynebacterium glutamicum, NCgl0581 promoter region, NCgl0580 (Genebank: NP_599841) promoter region, marker gene and plasmid backbone (such as figure 1 As shown, the expression of the transcriptional regulator Ncgl0581 is controlled by its own promoter Ncgl0581 promoter, while the expression of the marker gene GFP is controlled by the NCgl0580 promoter. The transcriptional regulator NCgl0581 can respond to the concentration of intracellular L-homoserine, thereby activating the expression of the NCgl0580 promot...

Embodiment 2

[0041] Example 2 Response test of L-homoserine biosensor to L-homoserine

[0042] The PhomBio plasmid described in Example 1 was electrotransformed into Corynebacterium glutamicum ATCC 13032 to obtain a recombinant bacterium containing L-homoserine biosensor. Pick a single colony and inoculate it into LBHIS liquid medium containing a final concentration of 25 ug / L kanamycin (2.5 g / L yeast extract; 5 g / L tryptone; 5 g / L sodium chloride; 18 g / L L brain heart infusion, 91 g / L sorbitol), 32°C, 200 rpm overnight culture for 16 h. The overnight culture solution was inoculated into fresh CGXII basic liquid medium containing 0-5 g / L L-homoserine at a final concentration of 0-5 g / L L-homoserine at 1% inoculum, and incubated at 32°C and 200 rpm for 6 h. Collect the cells by centrifugation, resuspend them with an equal volume of 0.9% NaCl, measure their fluorescence intensity with a multi-functional microplate reader, set the excitation wavelength to 488 nm and the emission wavelength t...

Embodiment 3

[0044] Example 3 Screening of L-homoserine high-yielding strains

[0045]The PhomBio plasmid described in Example 1 was electrotransfected into Corynebacterium glutamicum having a certain ability to synthesize L-homoserine to obtain a recombinant bacterium containing L-homoserine biosensor. A single colony was picked and inoculated into LBHIS liquid medium containing a final concentration of 25 ug / L kanamycin, and cultured overnight at 32°C and 200 rpm for 16 h. Using physical, chemical or biological methods, random mutagenesis is carried out on the above-mentioned recombinant strains to obtain a library of random mutant strains. In this example, the mutagenesis of the bacteria was carried out using the atmospheric pressure room temperature plasma mutagenesis instrument ARTP, and the specific steps were: wash the bacteria with physiological saline and resuspend to OD 600 = 1 to 2, take 10 μL of the bacterial solution and spread it on the patch, adjust the irradiation distance...

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Abstract

The invention provides a homoserine biosensor. The homoserine biosensor is constructed on the basis of a corynebacterium glutamicum transcription regulation factor NCgl0581, and a genome region containing an NCgl0581 open reading frame and an NCgl0580 promoter is cloned in front of GFP, so that the expression of the GFP is controlled by the NCgl0580 promoter. According to the sensor disclosed by the invention, the concentration of the L-homoserine is associated with the fluorescence signal intensity, so that the concentration of the L-homoserine is visualized, and the sensitive detection of the target product L-homoserine is realized. The biosensor can show a good linear relationship under the concentration of 0-5g / L of L-homoserine.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a biosensor and its construction method, and its application in screening high-yield amino acid strains. Background technique [0002] L-homoserine is a naturally occurring important functional amino acid. It is the precursor of methionine, lysine and threonine. It participates in various physiological and biochemical reactions and multiple biological metabolic processes in the body. function and application value. L-homoserine is used as an intermediate or raw material to synthesize a variety of chemical products, pesticides and feed, and is widely used in food, cosmetics, medicine and feed, and has broad application prospects. For example, L-homoserine is the main precursor raw material used to synthesize the new pesticide L-glufosinate, which has been widely used in foreign developed countries, and the demand is increasing year by year. [0003] At present, the synthesis methods...

Claims

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Application Information

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IPC IPC(8): C12N15/65C12N15/77G01N21/64C12R1/15
CPCC12N15/65C12N15/77G01N21/6486
Inventor 刘君魏亮赵金花徐宁
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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