Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for establishing lead ion sensitive whole-cell biosensor

A biosensor, lead ion technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of high detection limit and low sensitivity, and achieve the effect of improving sensitivity and reducing detection background value.

Inactive Publication Date: 2018-07-06
TIANJIN UNIV
View PDF9 Cites 9 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, a method for constructing a whole-cell biosensor with a gene cascade amplification expression system sensitive to lead ions is proposed; it overcomes the shortcomings of low sensitivity and high detection limit of the original biosensor

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for establishing lead ion sensitive whole-cell biosensor
  • Method for establishing lead ion sensitive whole-cell biosensor
  • Method for establishing lead ion sensitive whole-cell biosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction of recombinant expression vector pET28a-pbr-lux

[0039] Artificially synthesized lead ion-specific binding protein PbrR and bidirectional promoter P pbr , whose nucleotide sequence is shown in SEQ ID No.1; self-feedback regulatory protein LuxR, whose nucleotide sequence is shown in SEQ ID No.1. The nucleotide sequences shown in SEQ ID No.1 and SEQ ID No.4 were connected by enzyme-cut ligation, and the positive clones were screened to obtain the recombinant vector pET28a-pbr-lux. The plasmid map is shown in figure 1 shown.

[0040] The nucleotides shown in SEQ ID No.1 obtained through chemical total synthesis are respectively added with Bgl Ⅱ and BamH Ⅰ restriction sites at both ends of the fragment by PCR method, and FastDigest endonucleases Bgl Ⅱ and BamH Ⅰ are used for enzymatic digestion. Cutting, the reaction system is: 5 μL 10*FD buffer, 2.5 μL BglⅡ, 2.5 μL BamH Ⅰ, 30 μL nucleotide shown in SEQ ID No.1 with enzyme cutting site and 10 μL ul...

Embodiment 2

[0041] The nucleotide shown in SEQ ID No.4 in pGN68 was added with BamH Ⅰ and Xho Ⅰ restriction sites at both ends of the fragment by PCR method, and it was ligated with FastDigest endonuclease BamH Ⅰ and Xho Ⅰ The expression vector pET28a-pbr of the nucleotide shown in SEQ ID No.1 is subjected to double digestion, and then the nucleotide shown in SEQ ID No.4 is connected to the nucleoside shown in SEQ ID No.1 by DNA ligase On the expression vector pET28a-pbr of the acid, the enzyme digestion ligation reaction system and reaction conditions are according to the construction method of the expression vector pET28a-pbr connected with the nucleotide shown in SEQ ID No.1. After enzyme digestion and ligation, the competent cells E.coli DH5α were transformed, and positive clones were screened by colony PCR to obtain the recombinant vector pET28a-pbr-lux, which was verified by sequencing. Construction of embodiment 2 plasmid pGN68

[0042] The green fluorescent protein gene GFP conta...

Embodiment 3

[0044] Example 3 Recombinant expression vector pET28a-pbr-lux and plasmid pGN68 were co-transformed into Escherichia coli chassis strain E.coli DH5α (see figure 2 ).

[0045] The detailed construction steps of recombinant expression vector pET28a-pbr-lux and plasmid pGN68 co-transformed into E. coli chassis strain E.coliDH5α are as follows:

[0046] 1. Inoculate 100 μL of activated E. coli strain DH5α in 10 ml of LB medium, culture at 37°C, 220 rpm, until OD 600 When the temperature is 0.5-0.6, transfer to a 10ml centrifuge tube, centrifuge at 4500rpm / min in a pre-cooled 4°C centrifuge for 5min, remove the supernatant, and collect the bacteria;

[0047] 2. Wash the bacteria with 5ml of pre-cooled sterilized 0.1mol / L calcium chloride, centrifuge at 4500rpm / min for 5min in a pre-cooled 4°C centrifuge, remove the supernatant, collect the bacteria, and wash twice Second-rate;

[0048] 3. Pour off the supernatant as much as possible, add 50 μL 0.1mol / L calcium chloride, and 50 ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a lead ion sensitive whole-cell biosensor with a gene cascade amplification expression system and an establishment method. The establishment method comprises the following steps: connecting an artificial full-synthetic bidirectional promoter Ppbr and a lead ion specific binding protein PbrR such as a nucleotide sequence of SEQ ID No. 1 and a nucleotide sequence of SEQ ID No. 4 of a self-feedback regulation and control protein LuxR of plasmid pGN68 in a digestion mode, and screening positive cloning so as to obtain a target recombinant vector pET28a-pbr-lux; sequentially connecting a self-feedback promoter PluxR1, a green fluorescent protein nucleotide sequence and a self-feedback regulation and control protein LuxR nucleotide sequence of the plasmid pGN68 in the digestion mode; performing co-transformation on the recombinant vector and the plasmid pGN68 by using escherichia coli E.coli DH5alpha as a host, thereby obtaining the lead ion sensitive whole-cell biosensor.

Description

technical field [0001] The invention relates to the construction and use of a sensitive whole-cell biosensor capable of detecting the content of lead ions in an aqueous environment, which is used to quantify lead ions and provide help for heavy metal pollution restoration. Background technique [0002] Lead is a highly toxic substance needed by human beings. Car exhaust, smoke from plastic burning, paint, popcorn, preserved eggs and other foods all contain lead, which directly enters the human body through the human respiratory system, digestive system and even skin, and then Harmful to many organs of the human body, it can cause diseases such as lead encephalopathy, multiple neuritis and hemolytic anemia, and even lead to paralysis in severe cases. [0003] (1) Soil lead pollution: lead residues in soil mainly come from natural storage and human activities. Natural sources mainly refer to mineral resources. Man-made sources are mainly atmospheric deposition and industrial...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21G01N21/64C12R1/19
CPCC12N15/70G01N21/6486
Inventor 贾晓强卜蓉蓉刘一琳吴康
Owner TIANJIN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com