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Construction method for circuit amplification of cadmium ion whole-cell biosensor

A biosensor and cadmium ion technology, which is applied in the construction field of cadmium ion whole-cell biosensor circuit amplification, can solve the problems of low sensitivity and poor specificity, and achieve the effect of good sensitivity and specificity

Active Publication Date: 2021-04-30
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, poor specificity and low sensitivity have always been the bottleneck problems that limit the application of heavy metal microbial sensors.

Method used

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  • Construction method for circuit amplification of cadmium ion whole-cell biosensor
  • Construction method for circuit amplification of cadmium ion whole-cell biosensor
  • Construction method for circuit amplification of cadmium ion whole-cell biosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction of a whole-cell biosensor p2T7RNAPmut-68 capable of detecting cadmium ion content

[0040] 1. Plasmid construction (such as figure 1 shown)

[0041] Cadmium-specific promoter P cad SEQ ID No.1 and specific binding protein gene cadR sequence SEQ ID No.2 and T7RNAPmutSEQ ID No.4 (partial amino acid sequence such as figure 2 shown). In fragment cadR-P cad Add XhoⅠ and EcoRI restriction sites at both ends, and add XhoⅠ and EcoRI restriction sites at both ends of the fragment RBS-T7RNAPmut. T7 promoter P obtained by PCR amplification T7 SEQ ID No.5, cadO sequence SEQ ID No.6 and red fluorescent protein gene mcherrySEQ ID No.3. In fragment-P T7 -cadO-RBS-mcherry- add BamHI and EcoRI enzyme cutting sites at both ends respectively. Use FastDigest endonuclease for digestion, the reaction system 1) is: 5 μL 10*FD buffer, 2.5 μL XhoI, 2.5 μL EcoRI, 30 μL cadR-P with enzyme cutting site cad Fragment and 10 μL ultrapure water; reaction system 2) is: ...

Embodiment 2

[0050] Example 2 Performance Detection of Target Whole Cell Biosensor

[0051] 1. Growth curve of the target whole-cell biosensor

[0052] 1.1 Growth curve of target whole-cell biosensor chassis cells

[0053] A single colony of P. putida KT2440 containing the sensor plasmid was cultured overnight in LB medium containing antibiotics at 30 °C with a shaker at 220 rpm. Then diluted to OD with LB medium 600 1, inoculated into test tubes of LB liquid medium without resistance at a ratio of 1%; when the absorbance OD of the bacterial solution 600 When it is 0.6-0.8, add Cd with final concentration of 0, 0.1, 1, 10, 100, 200, 300, 400, 500, 600 μM to the test tube 2+ , and cultured on a shaker at 30°C and 220rpm. Cd not added 2+ Before, take a sample every 1 hour; add Cd 2+ After that, take a sample every 2 hours and measure the absorbance OD 600 . Wherein, absorbance is measured at a wavelength of 600 nm.

[0054] Data processing: Use the formula FIR=AFU / BFU to calculate t...

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Abstract

The invention relates to a construction method for circuit amplification of a cadmium ion whole-cell biosensor. According to the research, the cadmium whole-cell biosensor p2T7RNAPmut-68 is constructed in a mode of adding a T7RNAPmut (amino acid at the 40th site mutates into a termination codon) amplification module for the first time. The sensitivity and specificity of the biosensor are improved by amplifying a circuit through a T7RNAPmut (the 40th amino acid mutates into a termination codon) amplification module, so that the defects of a traditional cadmium ion biosensor in detection are overcome. Plasmid pCDFDuet-2 and pGN68 are used as carriers, and the cadmium ion whole-cell biosensor is obtained by utilizing a cadmium specific binding protein CadR, a reporter element mCherry, a cadO operon and a T7RNAPmut amplification module. The invention provides the construction method of a whole-cell biosensor sensitive to cadmium ions. The whole-cell biosensor sensitive to cadmium ions has wide adaptability.

Description

technical field [0001] The invention relates to a construction method for circuit amplification of a cadmium ion whole-cell biosensor; a method for circuit amplification of a cadmium ion whole-cell biosensor by using T7RNAPmut (the 40th amino acid is mutated into a stop codon), which is used for water Quantitative analysis of cadmium ions in the sample. Background technique [0002] As of 2020, the global population has increased to 7.5 billion, corresponding to the rapid advancement of industrialization. While rapid industrialization has continuously improved people's quality of life, it has also caused serious heavy metal pollution. Heavy metals are toxic, refractory, and easy to accumulate in the human body through the food chain, causing a series of health problems. On July 23, 2019, cadmium and cadmium compounds were included in the "List of Toxic and Harmful Water Pollutants (First Batch)". Cadmium pollution has been paid more and more attention by people. Therefore...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/78C12N15/65C12Q1/6897C12Q1/02C12R1/40
CPCC12N15/78C12N15/65C12Q1/6897C12Q1/025C12N2830/002G01N2333/21
Inventor 贾晓强刘腾马玉冰
Owner TIANJIN UNIV
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