A Microbiological Method for Detecting Heavy Metal Lead in Water

A technology for heavy metals and water bodies, which is applied to the establishment of a method for detecting lead in a water body environment and the construction of Escherichia coli, can solve the problems of high background detection results, unfavorable on-site detection, and low sensitivity, and achieves good effectiveness. , the result is stable and the effect of low fluorescence background value

Active Publication Date: 2019-05-10
WENZHOU MEDICAL UNIV
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  • Abstract
  • Description
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Problems solved by technology

[0006] The following problems exist in the construction of a microbial method that uses plasmids as carriers and bacteria as hosts to detect lead ions: (1) lead is a common highly toxic environmental pollution heavy metal, and most bacteria have a set of pumping mechanisms for harmful heavy metals. Resistant to lead deposits or active forms, so its sensitivity is not high
(2) The research is based on the plasmid as a fluorescent expression carrier. During the passage of the plasmid, there may be defects such as inconsistency in copy number, loss, and high background, resulting in unstable detection results
The detection of lead ions in Aparna needs to be cultivated in LB medium at 37°C for 12 hours. The cycle is too long and the timeliness is poor, which is not conducive to on-site detection.

Method used

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  • A Microbiological Method for Detecting Heavy Metal Lead in Water
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  • A Microbiological Method for Detecting Heavy Metal Lead in Water

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Construction of double knockout sensitive strains, see figure 1

[0052] 1.1 PCR amplification of chloramphenicol resistance gene containing zntA and zntR upstream and downstream 50bp homologous arms with FRT sites

[0053] According to the zntA / zntR gene sequence published by http: / / ecogene.org / and the cat gene sequence published by GenBank, a chloramphenicol resistance gene containing zntA and zntR upstream and downstream 50bp homologous arms with FRT sites was designed and synthesized and its identification primers. The specific information is shown in Table 1. The primers were synthesized by Shanghai Jierui Bioengineering Co., Ltd. with sterile ddH 2 O Dissolve the primers, prepare a storage solution with a concentration of 10 μmol / L, and store at -20°C.

[0054] Table 1. Gene Knockout Primer Details Table

[0055]

[0056] Take 1ml of MC4100 overnight bacterial solution containing plasmid pKD3 in a 1.5ml EP tube, centrifuge at 12,000rpm for 1min...

Embodiment 2

[0068] Example 2. The fusion of gene knock-in fragments, such as figure 2

[0069] 2.1 Primer information and synthesis

[0070] According to Harvard MoLecuLar TechnoLogy Group&Lipper Center for ComputationaL Genetics website http: / / arep.med.harvard.edu / Labgc / adnan / projects / EcoLiKOprimers / EcoLiKOprimers.htm provided primer sequence knockout zntA gene, http: / / ecogene.org / The published zntA gene sequence and GenBank published PpbrA-pbrR-PpbrA::DsRed-express2 gene sequence, designed the internal primers (P 2 : zntA-Ni and P 5 : zntA-Ci) and outer primer (P 1 :zntA-No and P 6 :zntA-Co), make P 2 with P 3 , P 4 with P 5 There is a complementary sequence between them, and the two outer primers of gene knockout remain unchanged, P 3 with P 4 A pair of primers for amplifying the PpbrA-pbrR-PpbrA::DsRed-express2 gene. The specific information is shown in Table 2. The primers were synthesized by Shanghai Jierui Bioengineering Co., Ltd. with sterile ddH 2 O Dissolve the pr...

Embodiment 3

[0091] Example 3. Sequencing of gene knock-in fragments

[0092] 3.1 Gene knock-in fragment plus A reaction and purification: The PCR reaction system is as follows

[0093]

[0094] The PCR reaction conditions are as follows: 72°C, 1h, 4°C, 30min. 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.

[0095] 3.2 The target fragment is connected to the pMD19-T simple vector

[0096] According to the instructions of the pMD19-T simpLe Vector kit from TaKaRa Company, the T vector was connected to the target gene, and the connection reaction system was as follows:

[0097]

[0098] 16°C, ligate overnight.

[0099] 3.3 Conversion

[0100] by cold CaCl 2 The ligation product was transformed into Escherichia coli DH5α competent cells.

[0101] 3.4 Sequencing

[0102] Pick an outer primer P 1 and P 6 The correct monoclonal strains identified by PCR and plasmid digestion were se...

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Abstract

The invention provides a construction method of an Escherichia coli engineering strain for detecting heavy metal lead and a method for detecting lead in a water body. The gene engineering strain provided by the invention is PpbrA-pbrR-PpbrA:DsRed-express2 / E.colideltazntRdeltazntA named as E.coli WMC-008p CGMCC No.9748, wherein zntA and zntR genes are subjected to deletion mutation and inactivation, and a pbr operon from a cupriavidus metallidurans CH34 strain is input to express a red fluorescence protein DsRed-express2. Compared with an Escherichia coli reporter strain containing a reporter plasmid, the engineering strain provided by the invention has the advantages of simplicity and convenience in operation, low fluorescence background value, stable signal and the like. The engineering strain provided by the invention can reflect the bioavailability of lead in the water body and can provide the basis for objectively evaluating the biotoxicity effect of lead in the water body.

Description

technical field [0001] The invention relates to a microbiological method for detecting heavy metal lead, in particular to a construction method of Escherichia coli transformed by genetic engineering technology and establishment of a method for detecting lead in water environment. Background technique [0002] With the development of industry and agriculture, many pollutants containing lead ions are discharged into rivers and lakes, polluting soil, water sources and even daily food and vegetables. Lead in the environment mainly comes from various paints, coatings, batteries, smelting, hardware, machinery, electroplating, cosmetics, hair dyes, glazed dishes, tableware, coal, puffed food, water pipes, etc. Studies have found that excessive heavy metal lead is toxic to all living organisms. After the human body absorbs excessive lead, it can accumulate in the vital organs of the human body such as the liver, kidney, and brain. It enters the body through the skin, digestive trac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C12Q1/689C12Q1/02C12R1/19
Inventor 吕建新严锡娟叶薇李江辉
Owner WENZHOU MEDICAL UNIV
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