Construction of sensitive arsenic ion whole cell biosensor and arsenic ion concentration detection method

A technology for biosensors and construction methods, applied in the field of construction of arsenic ion whole-cell biosensors, can solve the problems of low sensitivity and poor specificity, and achieve the effects of wide adaptability, high sensitivity and specificity

Pending Publication Date: 2020-04-14
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a method for constructing a whole-cell biosensor sensitive to arsenic ions and having a gene cascade ampl

Method used

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  • Construction of sensitive arsenic ion whole cell biosensor and arsenic ion concentration detection method
  • Construction of sensitive arsenic ion whole cell biosensor and arsenic ion concentration detection method
  • Construction of sensitive arsenic ion whole cell biosensor and arsenic ion concentration detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1 Construction of a sensitive whole-cell biosensor capable of detecting the content of arsenic ions As(Ⅲ)

[0037] 1. Construction of detection plasmid pCDF-As-luxR (attached figure 1 shown)

[0038] Arsenic-specific promoter P amplified by PCR ars (SEQ ID No.1) and the nucleotide shown in the specific binding protein gene arsR sequence (SEQ ID No.2), i.e. fragment P ars -arsR. in fragment P ars BamHI and EcoRI restriction sites were added to both ends of -arsR respectively. Use FastDigest endonuclease for digestion, the reaction system is: 5 μL 10*FD buffer, 2.5 μL BamHI, 2.5 μL EcoRI, 30 μL P ars -arsR fragment and 10 μL of ultrapure water. The reaction conditions are: 37°C, 2h. Using a PCR purification kit, add 250 μL of Bingding Buffer solution to 50 μL of the digested product, mix well, add to the adsorption column, let stand for one minute, centrifuge at 10,000xg for 1 minute, and discard the effluent. Add 650μL Wash Buffer, centrifuge at 10,000g ...

Embodiment 2

[0050] Example 2 Detection of arsenic ion As(Ⅲ) by target whole-cell biosensor

[0051] 1. Fermentation of target whole-cell biosensor chassis cells and fluorescence detection induced by arsenic standard solution

[0052] The target whole-cell biosensor chassis cells were inoculated in LB culture-based test tubes according to the inoculum amount of 1%, and cultured overnight at 37° C. and 220 rpm for 14 hours to obtain a seed culture solution. Take the seed culture solution according to 1% inoculum amount, transfer it to 5 mL non-resistant LB medium test tubes, add arsenic ions As(Ⅲ) to different test tubes respectively, so that the arsenic ions As(Ⅲ) in each test tube The final concentrations were 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, and 200 μM, and three parallel samples were set for each concentration of arsenic ion As(Ⅲ), and incubated at 37°C and 220rpm for 8h .

[0053] Samples were taken for absorbance and fluorescence signal detection. The absorbance is measu...

Embodiment 3

[0058] Example 3 Detection of arsenic ion As(Ⅲ) by target whole-cell biosensor

[0059] The target whole-cell biosensor chassis cells were inoculated in a test tube containing LB medium according to a 2% inoculation amount, and cultured overnight at 37° C. at 220 rpm for 12 hours to obtain a seed culture solution. Take the seed culture solution according to 2% inoculum amount, transfer it to LB medium test tubes containing 5 mL of non-resistance, and add arsenic ions As(Ⅲ) to different test tubes respectively, so that the arsenic ions As(Ⅲ) in each test tube The final concentrations were 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, and 200 μM, and three parallel samples were set for each concentration of arsenic ion As(Ⅲ), and incubated at 37°C and 220rpm for 8h .

[0060] The remaining experimental steps and data processing are the same as in Example 2, and the experimental results are also the same as in Example 2.

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Abstract

The invention relates to construction of a sensitive arsenic ion whole cell biosensor and an arsenic ion concentration detection method. The sensor detection plasmid and the reporter plasmid are recombinant. The detection plasmid consists of an arsenic-specific protein regulatory promoter Pars, an arsenic-specific binding protein ArsR gene, and a self-feedback regulatory protein gene luxR. The reporter plasmid consists of a self-feedback promoter PluxRI, a red fluorescent protein mCherry gene and a self-feedback regulatory protein gene LuxR. By introducing a positive feedback amplification system based on the variant LuxR protein as a regulatory element, the sensitivity of the sensor is increased by 20 times to 0.1 micron, and the specific signal ratio is increased by 1.5 to 7.5 times. Thearsenic ion biosensor takes Escherichia coli DH5 as a host cell, has high sensitivity and specificity for arsenic ion detection, is not interfered by other metal ions, and has wide adaptability.

Description

technical field [0001] The invention relates to the construction and use of a sensitive whole-cell biosensor capable of detecting the content of arsenic ions As(Ⅲ), which is used for quantitative analysis of arsenic ions in water samples. In particular, it relates to the construction of a sensitive arsenic ion whole-cell biosensor and a method for detecting the arsenic ion concentration. Background technique [0002] Arsenic (As) is a metalloid element with both physical and chemical properties of metals and non-metals. As one of the most common environmental poisons, it widely exists in water, soil, air and food in different forms of compounds, and is listed as the first-class harmful substance by the US Environmental Protection Organization. [0003] Arsenide in the environment mainly exists in two forms: arsenite As(Ⅲ) and arsenate As(V). In these two forms, the toxicity of arsenite As(Ⅲ) is the (V) 20-60 times, is listed as the first class of carcinogens. Ingestion of...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/65C12Q1/6897C12Q1/02C12R1/19
CPCC12N15/70C12N15/65C12Q1/6897C12Q1/025G01N2333/245
Inventor 贾晓强马玉冰卜蓉蓉
Owner TIANJIN UNIV
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