A strain of Escherichia coli that detects arsenic

A technology of Escherichia coli and escherichiacoli, which is applied in the direction of bacteria, microbial-based methods, and microbial detection/testing, can solve the problems of slow development of heavy metal microbial detection technology, and achieve easy promotion, stable detection signals, and sample pre-treatment. Handle simple effects

Active Publication Date: 2019-08-02
WENZHOU MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The application of heavy metal microbial detection technology is limited and the development is slow

Method used

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  • A strain of Escherichia coli that detects arsenic
  • A strain of Escherichia coli that detects arsenic
  • A strain of Escherichia coli that detects arsenic

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1. Preparation of ΔarsB mutant bacteria

[0066] 1.1 Primer information and synthesis

[0067] Knockout Primers: According to http: / / ecogene.org / Provide primer sequences for gene knockout, Primer5.0 and DNAMAN software, design homologous recombination primers (see Table 1), the 5' end is the homology arms on both sides of the arsB gene, and the 3' end is used to amplify chloramphenicol resistance gene. upstream homology arm primer H 1 -P 1 ; Downstream homology arm primer H 2 -P 2 .

[0068] Gene knockout identification primers: using Harvard Molecular Technology Group&Lipper Center for Computational Genetics website

[0069] http: / / arep.med.harvard.edu / labgc / adnan / projects / EcoliKO-primers / EcoliKOprimers.htm The provided pair of knockout identification primers designed spanning the outside of the gene to be knocked out (see Table 2). Identification of arsB gene knockout primer design: the upstream primer arsB-jianding-F is located 108bp upstrea...

Embodiment 2

[0102] Embodiment 2. Construction of fusion reporter gene pars-arsR-gfpmut2

[0103] 2.1 Primer information and synthesis

[0104] According to the target gene pars and arsR sequences published by GenBank and the known gfpmut2 gene sequence, use the Primer5.0 software to design primers (see Table 5), and some primers introduce restriction sites at the 5' end or 3' end of the gene. Pars-arsR gene 5′ end primer (P 3 : pars-arsR-F) and 3' end primer (P 4 :pars-arsR-R), make P 4 with P 5 Complementary sequences exist between, P 5 (gfpmut2-F) and P 6 (gfpmut2-R) is a pair of primers for amplifying the gfpmut2 gene, and M13F and M13R are primers for identifying the pars-arsR-gfpmut2 fragment on the pMD19-T vector. Design the inner primers (P 7 : araB-Ni and P 8 : araB-Ci) and outer primer (P 9 :araB-No and P 10 : araB-Co), gene knockout two outer primers unchanged, P 7 with P 8 A pair of gene primers for amplifying pars-arsR-gfpmut2, P 11 with P 12 A pair of gene prime...

Embodiment 3

[0140] Example 3 Gene knock-in

[0141] 3.1 Fusion of gene knock-in fragments

[0142] 3.1.1 Amplification of two homology arms:

[0143] Take 1mL of the overnight bacterial solution of E.coli MC4100 in a 1.5mL EP tube, centrifuge at 8000rpm for 3min, discard the supernatant, and use 1mL MilliQ H 2 After O was resuspended and washed twice, boiled in water for 10 min, centrifuged at 12000 rpm for 3 min, and 24 μL of the supernatant was taken as a template. Make a system according to the table below, and do PCR identification.

[0144]

[0145] The PCR reaction conditions were as follows: 94°C, 1min; 88°C, 4min; 94°C, 10sec, 66°C, 3min, 25 cycles; 72°C, 10min, 4°C storage. 5 μL of the product was identified by 1.0% Agarose gel electrophoresis. Purify the PCR product with a purification kit, and finally add an appropriate amount of sterile MilliQ H 2 O dissolved and eluted for later use.

[0146] 3.1.2 Amplification of the pars-arsR-gfpmut2 gene:

[0147] Take 1 mL of t...

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Abstract

The invention relates to a microorganism method for detecting metallic-arsenic-like in water, in particular to an escherichia coli report bacterial strain culturing method improved through genetic engineering, and an escherichia coli culturing method for detecting arsenic in water. The detection bacterial strain is obtained by adopting a Red recombination system to knockout arsenic resistant arsB genes of wild type escherichia coli MC4100, and then adopting the gene knockin technology to substitute pars-arsR-gfpmut2 report genes to the positions of araB coding genes. The biological detection bacterial strain is named as E.coli WMC-011p. The lowest arsenic detection range meets the Integrated Wastewater Discharge Standard of GB8978-1996. The bacterial strain overcomes the shortcomings of high fluorescence background value, unstable detection signals, inaccurate results and the like of a biological detection bacterial strain based on a plasmid vector, and has the advantages of high specificity, high sensitivity, low cost and the like.

Description

technical field [0001] The invention relates to a microbiological method for detecting metalloid arsenic in water body, in particular to a method for constructing a reporter strain of Escherichia coli transformed by genetic engineering, and a method for establishing the same for detecting metalloid arsenic in water body environment. Background technique [0002] Arsenic is a non-metallic element widely distributed in nature. The content in the earth's crust is about 2 to 5 mg / kg, which is the 20th element that constitutes the earth's crust. Metalloid arsenic is classified as a Class I human carcinogen by the International Agency for Research on Cancer (IARC). Twenty-one countries in different parts of the world have been affected by arsenic pollution. The largest risk group is from Bangladesh, followed by West Bengal in India. China is also one of the countries with the most serious arsenic hazards. Metal-like arsenic pollution in water bodies has been manifested as public...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12Q1/6897C12Q1/02C12R1/19
Inventor 吕建新纪松军周怀彬杜璟郑美琴
Owner WENZHOU MEDICAL UNIV
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