Microbiological method for detecting heavy-metal cadmium in water body

A water body and wild-type technology, applied to the construction of Escherichia coli, established in the field of detection of heavy metal cadmium in the water environment, can solve the problems of engineering strains without literature reports, achieve reliable results, reduce background values, good repeatability

Active Publication Date: 2015-09-30
WENZHOU MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The engineering strains constructed in this study have not bee

Method used

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  • Microbiological method for detecting heavy-metal cadmium in water body
  • Microbiological method for detecting heavy-metal cadmium in water body
  • Microbiological method for detecting heavy-metal cadmium in water body

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1. Preparation of mutant bacteria

[0068] 1. Primer information and synthesis

[0069] Gene knockout primers: According to the gene knockout primer sequence provided by http: / / ecogene.org / , Primer5.0 and DNAMAN software, design homologous recombination primers, see Table 1, the 5' end is the homology on both sides of the cat gene For the arm, see the ununderlined part in Table 1, and the 3' end is used to amplify the chloramphenicol resistance gene, see the underlined part in Table 1. Upstream homology arm primer zntA-Nf, sequence as shown in SEQ ID NO.6 and zntR-Nf, sequence as shown in SEQ ID NO.8; downstream homology arm primer zntA-Cr, sequence as shown in SEQ ID NO.7 Shown and zntR-Cr, the sequence is shown in SEQ ID NO.9.

[0070] Gene knockout identification primers: use a pair spanning the gene to be knocked out provided by Harvard Molecular Technology Group & Lipper Center for Computational Genetics website http: / / arep.med.harvard.edu / labgc / adnan / ...

Embodiment 2

[0117] Embodiment 2. Construction of fusion reporter gene pmerR-MerR(M)-pmerR-gfpmut2

[0118] 1. Primer information and synthesis

[0119] According to the target gene pmerR and MerR(M) sequences published by GenBank and the known gfpmut2 gene sequence, use the Primer5.0 software to design primers, see Table 7, some primers introduce restriction sites at the 5' end or 3' end of the gene , design the primer Pmer-MerR-m-F for amplifying pmerR-MerR (M), the sequence is shown in SEQ ID NO.20 and Pmer-MerR-m-R, the sequence is shown in SEQ ID NO.21, amplify the pmerR-gfpmut2 gene The primer Pmer-GFPmut2-F sequence is shown in SEQ ID NO.22 and the GFPmut2-R sequence is shown in SEQ ID NO.23, and the final fusion fragment is connected to the identification primer M13F of the plasmid vector pMD19-T, and the sequence is shown in SEQ ID As shown in NO.24 and M13R, the sequence is shown in SEQ ID NO.25. See Table 7 for specific information, and the primers were synthesized by Invitrog...

Embodiment 3

[0158] Example 3. Gene knock-in

[0159] 1. Primer information and synthesis

[0160] Use Primer5.0 software to design primers, see Table 14, design pmerR-MerR(M)-pmerR-gfpmut2 gene N-terminal and C-terminal inner primers, P 2 : zntR-cd-Ni, sequence as shown in SEQ ID N0.15 and P 5 : zntR-cd-gfpmut2-Ci, the sequence is shown in SEQ ID NO.16 and the outer primer, P 1 : zntR-No, the sequence is shown in SEQ ID N0.14 and P 6 : zntR-Co, the sequence is as shown in SEQ ID N0.17, making P 2 : zntR-cd-Ni with P 3 : pmerR-MerR(M)-pmerR-gfpmut2-F, the sequence is shown in SEQ ID NO.18, P 4 : pmerR-MerR(M)-pmerR-gfpmut2-R, the sequence is shown in SEQ ID N0.19, and P s : There is a complementary sequence between zmR-cd-gfpmut2-Ci, the gene knockout two outer primers are unchanged, P 3 with P 4 A pair of gene primers for amplifying pmerR-MerR(M)-pmerR-gfpmut2, P 7 : pkov-SalI, sequence as shown in SEQ ID N0.28 and P 8 : pkov-NotI, the sequence is as shown in SEQ ID No. 29, whic...

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Abstract

The invention relates to a microbiological method for detecting heavy-metal cadmium, particularly a construction method of an Escherichia coli engineering strain for detecting cadmium and a method for detecting cadmium in water by using the Escherichia coli engineering strain. The construction method of the engineering strain comprises the following steps: knocking out Escherichia coli cadmium-resistant zntA and zntR genes by using a Red recombination system, and substituting a pmerR-MerR(M)-pmerR-gfpmut2 report gene to the zntR coding gene position by using a gene knock-in technique. The lowest detection range of the engineering strain for cadmium conforms to Sewage Comprehensive Discharge Standard-GB8978-1996. The engineering strain overcomes the defects of high fluorescent background level, poor detection signal and the like in the plasmid-vector-based engineering strain. The engineering strain provided by the invention can be complementary with AAS (atomic absorption spectroscopy) and other physicochemical analysis processes to analyze the biological availability and total amount of the cadmium, thereby providing references for objective evaluation on biological toxicity of the cadmium.

Description

technical field [0001] The invention relates to a microbiological method for detecting heavy metal cadmium in water, in particular to a method for constructing Escherichia coli transformed by genetic engineering technology, and a method for establishing the method for detecting heavy metal cadmium in water environment. Background technique [0002] The heavy metal cadmium (Cadmium, Cd) is classified as the first human carcinogen by the International Agency for Research on Cancer (IARC); the US National Toxicology Program (NTP) also recognizes cadmium as a human carcinogen. Heavy metal cadmium pollution in water has manifested as public hazards in some areas of our country, such as the cadmium rice incident in Guangdong and the cadmium pollution incident in Longjiang, Guangxi, which have aroused people's great attention. Cadmium is very stable in the environment and is not easy to be biodegraded. The content in water and soil increases year by year, and then accumulates in so...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12Q1/68C12Q1/02C12R1/19
Inventor 吕建新吕攀攀王伍卢彬彬林炜炜
Owner WENZHOU MEDICAL UNIV
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