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Biosensor capable of responding to 3-dehydroshikimic acid and application of biosensor

A biosensor, dehydroshikimic acid technology, applied in DNA/RNA fragments, recombinant DNA technology, bacteria, etc., can solve the problem of unreported positive correlation between cusR and 3-dehydroshikimic acid, etc.

Active Publication Date: 2018-05-01
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, the published information has never reported a positive correlation between cusR and 3-dehydroshikimic acid.

Method used

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  • Biosensor capable of responding to 3-dehydroshikimic acid and application of biosensor
  • Biosensor capable of responding to 3-dehydroshikimic acid and application of biosensor
  • Biosensor capable of responding to 3-dehydroshikimic acid and application of biosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Transcriptome analysis mining 3-dehydroshikimic acid response function regulatory genes

[0065] Take the 3-dehydroshikimic acid-producing strain E.coli WJ060 as the experimental strain and the non-3-dehydroshikimic acid-producing strain E.coli ATCC 8739 as the control strain, pick a single colony and inoculate the LB medium, shake the flask at 37°C Fermentation 24h, bacterial strain E.coli WJ060 selects the fermentation medium (WJ060-12h and WJ060-20h) of two sampling points of fermentation culture 12h and 20h, bacterial strain E.coli ATCC 8739 selects the fermentation medium of fermentation culture 12h sampling point (8739 -12h). The fermentation broth was centrifuged at 4°C and 3200rpm for 5 minutes, and the bacterial cells were quickly frozen in liquid nitrogen and sent for transcriptome sequencing analysis.

[0066] In order to mine 3-dehydroshikimate-responsive functional genes, the bioinformatics software Bowtie2 was used to compare the three transcri...

Embodiment 2

[0072] Example 2: Real-time PCR verification of transcriptional regulatory genes responding to 3-dehydroshikimic acid

[0073] 1) Real-time PCR verification of transcriptional regulatory genes in response to endogenous production of 3-dehydroshikimate

[0074] The 3-dehydroshikimate-producing strain E.coli WJ060 was used as the experimental strain, and a single colony was picked and cultured in LB medium at 37°C overnight. Then transfer the NBS medium with 2% inoculum of 2% overnight culture solution, shake the flask for fermentation and culture at 37°C, take samples at four time points of 12h, 15h, 21h, and 24h, and control the 10 cells according to the real-time PCR operation process. Genes were verified by real-time PCR, and the responses of 10 transcriptional regulatory genes to the continuous accumulation of intracellular 3-dehydroshikimic acid product concentration during fermentation were analyzed.

[0075] The result is as figure 2 , at the four fermentation time po...

Embodiment 3

[0081] Example 3: Construction of 3-dehydroshikimic acid biosensor

[0082] In order to construct a 3-dehydroshikimate-sensing biosensor, it is first necessary to obtain a DNA fragment of the 3-dehydroshikimate-responsive functional fusion element, which includes the regulatory gene cusR, the fluorescent reporter gene gfp, and the linker sequence L (Linker) part. Among them, the intermediate connection sequence L connecting the functional response regulatory gene cusR and the gfp reporter gene is designed as a short peptide of 15 amino acids, and the coding sequence of the gfp gene without the start codon and the coding sequence of the positively related gene cusR without the stop codon The coding sequence of L is 5'-ggtggtggtggttctggtggtggtggatccggtggcggtggttct-3.

[0083] In order to obtain the cusR gene fragment of the regulatory gene, this experiment used the genome of the strain E.coli ATCC 8739 as a template, and used the upstream primer sequence: 5'-ccggaattcggctgagtga...

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Abstract

The invention achieves success in constructing a strain for producing 3-dehydroshikimic acid (DHS) via such means as genetic engineering or metabolic engineering, but the large-scale production of the3-dehydroshikimic acid via biological means faces the biggest bottleneck in the rapid acquisition of an efficient and stable-yield strain. The biosensor provided by the invention consists of a regulatory gene cusR and / or a promoter of the regulatory gene cusR and a reporter gene; positively correlated response to concentration of the 3-dehydroshikimic acid is achieved; and a technical bottleneckthat a 3-dehydroshikimic acid compound, which is colorless and is in lack of an effective chromogenic reaction, is difficult in achieving high-throughput screening; therefore, a strain mutagenesis library screening method for producing the 3-dehydroshikimic acid is further developed out.

Description

technical field [0001] The invention relates to the fields of biosensor and biotechnology, in particular to a novel 3-dehydroshikimic acid responsive biosensor, a transcription expression system comprising the biosensor and a high-throughput screening system comprising the biosensor. Background technique [0002] 3-dehydroshikimate (3-dehydroshikimate, DHS) is an intermediate metabolite necessary for the survival of plants and microorganisms. After further processing, it can form high value-added products. It is an important raw material for pharmaceuticals and fine chemicals. The traditional chemical method is used for synthesis, because benzene or toluene is used as the raw material for its synthesis, which often causes harm to the human body and the environment. In recent years, the use of genetic engineering or metabolic engineering to produce 3-dehydroshikimic acid has been successful, but the rapid and stable production of strains has become the biggest bottleneck in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N1/21
CPCC12N1/20C12N15/11
Inventor 王钦宏涂然李梁坡李林陈五九
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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