Microbiology method for detecting metallic arsenic in water body

A technology for metal arsenic and water body is applied in the field of microbiology for detecting metal arsenic in water body, which can solve the problems of slow development of heavy metal microbial detection technology, and achieve the effects of easy promotion, reliable results and stable detection signals.

Active Publication Date: 2015-09-16
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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  • Microbiology method for detecting metallic arsenic in water body
  • Microbiology method for detecting metallic arsenic in water body
  • Microbiology method for detecting metallic arsenic in water body

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] Knockout Identification Primers: Using the 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 upstream ...

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 sequence 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 s (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 primer...

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 12 000 rpm for 3 min, and 24 μL of the supernatant was taken as a template. According to the table dubbed system, 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 the overni...

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Abstract

The invention relates to a microbiology method for detecting metallic arsenic in water body, which more specifically relates to an Escherichia coli report bacterial strain construction method through gene engineering reconstruction, and relates to a method for establishing arsenic in water body. According to the construction of detection bacterial strain, a Red recombination system is employed for deleting wild type escherichia coli MC4100 arsenic resistance arsB gene, and then a gene integration technology is employed for displacing pars-arsR-gfpmut2 reporter gene to the araB coding gene position. The biological detection bacterial strain is named as E.coli WMC-011p, and a lowest detection scope for arsenic accords with a sewage integration discharge standard-GB8978-1996. The bacterial strain overcomes the disadvantages of high fluorescence background value, unstable detection signal and inaccurate result of the biological detection bacterial strain based on a plasmid carrier, and has the characteristics of strong specificity, high sensitivity and low cost.

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 Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12Q1/68C12Q1/02C12R1/19
Inventor 吕建新纪松军周怀彬杜璟郑美琴
Owner WENZHOU MEDICAL UNIV
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