Construction method of microbial sensor and application method of microbial sensor

A technology of a microbial sensor and a construction method, applied in the field of microbial sensor construction, can solve problems such as quantitative detection that have not yet been seen, and achieve the effects of reducing detection difficulty, high sensitivity, and improving sensitivity

Pending Publication Date: 2019-07-12
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Regulatory genes / promoters are required to have a specific response to the target in order to enhance expression or activation, and most of the biomarkers with clinical detection significance are macromolecular proteins, which cannot directly enter the microorganisms. Therefore, microbial sensors are mostly used for small molecules The quantitative detection of substances such as heavy metal ions, amino acids, etc., has not been reported for the quantitative detection of proteins and other macromolecules

Method used

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  • Construction method of microbial sensor and application method of microbial sensor
  • Construction method of microbial sensor and application method of microbial sensor
  • Construction method of microbial sensor and application method of microbial sensor

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Embodiment 1

[0044] figure 1 It is the schematic diagram of the preparation circuit of the microbial sensor in Example 1 of the present invention, such as figure 1 Shown, the invention provides a kind of construction method of microbial sensor, comprises the following steps:

[0045] Step 1 constructing a deficient strain of the amino acid to be tested;

[0046] Step 2 Construct a plasmid with a fluorescent protein gene as a reporter gene, intercept the target gene fragment required for the LuxI / LuxR cyclic amplification circuit, connect and construct, obtain a plasmid containing a cyclic amplification circuit based on the LuxI / LuxR system, and transform the recombinant plasmid into a competent state In the bacterial strain obtained in step 1, the reporter gene fragment is repeatedly expressed to obtain a microbial sensor with signal cycle amplification capability;

[0047] Step 3: Cultivate the strain obtained in Step 2 to the logarithmic growth phase, and perform starvation culture in ...

Embodiment 2

[0058] The preparation method of the microbial sensor with the function of circularly amplifying the signal takes lysine as the amino acid to be detected and GFP as the reporter gene.

[0059] Step 1: Construction of lysine-deficient Escherichia coli MG1655

[0060] (1) Select the 50 bp sequences on both sides of the lysA gene to be knocked out as the upstream and downstream homologous recombination arms of gene targeting. The upper and lower homologous recombination arms are designed to be located on both sides of the specific primers for the kanamycin resistance gene (kan), and long primers that can be used for PCR amplification are obtained by chemical synthesis.

[0061] (2) Using the plasmid pKD4 as a template, the lysA gene targeting fragment was obtained by high-fidelity PCR amplification with the above-mentioned long primers.

[0062] (3) Preparation of electroporation competent cells of MG1655 strain. The pKD46 helper plasmid of the Lambda Red recombination system w...

Embodiment 3

[0092] In the present invention, the amino acid to be tested is a monomeric amino acid or a polypeptide. Taking lysine as an example, a lysine polypeptide is used because the polypeptide can be hydrolyzed into multiple lysine molecules, and it is easier to make lysine Acid-deficient E. coli grows, making it easier to scale up the assay. The polypeptide sequences used are CKKKKK for CK5, CKKKKKKKKKK for CK10, and CKKKKKKKKKKKKKKK for CK15.

[0093] The microbial sensor is starvation-treated lysine-deficient Escherichia coli with gfp as the reporter gene, and the original lysine molecules have been depleted. At this time, different groups of lysine polypeptide molecules with different concentrations are added to the M9 medium. The amplification response of lysine polypeptide molecules relative to lysine can be determined, and the specific steps are as follows:

[0094] (1) Prepare three kinds of lysine polypeptide solutions of CK5, CK10 and CK15 with deionized water, filter and...

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Abstract

The invention provides a construction method of a microbial sensor and an application method of a microbial sensor. The construction method comprises the steps of: the step 1, constructing an amino acid defective strain to be detected; the step 2, intercepting target gene fragments required by an LuxI/LuxR circulating amplification line for connection to obtain a plasmid comprising the circulatingamplification line based on an LuxI/LuxR system, and transforming the recombinant plasmid into an amino acid defective strain to ensure that the report gene fragments are repeatedly expressed; and the step 3, culturing the strain to a logarithmic growth phase, and performing starvation culture in a culture medium. According to the invention, the amino acid defective strain to be detected is circularly amplified and is used as a microbial sensor, the nucleic acid aptamer combined with the protein marker and having high specificity and high affinity with a protein marker is used as a 'bridge',the detection of the protein marker is converted into the detection of the amino acid to be detected, the constructed microbial sensor is used for achieving the detection of the macromolecular biomarker, and a circular amplification system is combined as a signal amplification means so that the sensitivity of quantitative detection is greatly improved.

Description

technical field [0001] The invention belongs to the field of biomedical detection, and in particular relates to a construction method of a microbial sensor and an application method thereof. Background technique [0002] Biomarkers are molecules that undergo significant changes during the course of disease, which can be nucleic acids, proteins, metabolites, isoenzymes, or hormones, etc., and have significance in disease early warning, disease diagnosis, and disease prognosis. Biomarkers can objectively measure and evaluate normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions. At present, the methods for quantitative detection of biomarkers include enzyme-linked immunoassay (ELISA), chemiluminescence immunoassay, and fluorescent immunoassay. Effective detection is difficult to meet the needs of clinical diagnosis for highly sensitive detection of biomarkers. [0003] Microbial sensors use microorganisms as sensitive se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N21/64
CPCG01N33/533G01N21/6486
Inventor 吕雪飞张静邓玉林陶慧杨元展
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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