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A strain of Escherichia coli detecting cadmium

A technology of Escherichia coli and escherichiacoli, which is applied in the direction of bacteria, microbe-based methods, and microbiological determination/testing, can solve the problems of no literature reports on engineering strains, and achieve reliable results, good repeatability, and detection signals stable effect

Active Publication Date: 2019-01-25
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The engineering strains constructed in this study have not been reported in the literature at home and abroad after searching

Method used

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  • A strain of Escherichia coli detecting cadmium
  • A strain of Escherichia coli detecting cadmium
  • A strain of Escherichia coli detecting cadmium

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 of primers spanning the gene to be knocked out provided by Harvard Molecular Technology Group & Lipper Center for Computational Genetics website http: / / arep.med.harvard.edu / l...

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 the Primer5.0 software to send out the primers, see Table 14, design the inner primers of the pmerR-MerR(M)-pmerR-gfpmut2 gene N-terminal and C-terminal, P 2 : zntR-cd-Ni, the sequence is shown in SEQ ID NO.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 NO.14 and P 6 : zntR-Co, the sequence is shown in SEQ ID NO.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 SEQID NO.19, and P 5 : There is a complementary sequence between zntR-cd-gfpmut2-Ci, the gene knockout two outer primers remain 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 NO.28 and P 8 : pkov-NotI, the sequence is as sho...

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Abstract

The present invention relates to a microbiological method for detection of heavy metal cadmium, and in particular relates to a construction method of an escherichia coli engineered strain for detection of cadmium and construction of a method of for detection of cadmium in water. The construction method of the escherichia coli engineered strain is as follows: first, escherichia coli cadmium resistant zntA and zntR genes are knocked out by use of Red recombination system, then pmerR-MerR (M)-pmerR-gfpmut2 reporter gene is replaced to the position of zntR encoding gene location by gene knockin technology to obtain the escherichia coli engineered strain. The minimum cadmium detection range of the escherichia coli engineered strain is in line with Integrated Wastewater Discharge Standard-GB8978-1996, and the escherichia coli engineered strain overcomes the defects that a plasmid vector based engineered strain has high fluorescent background value, poor detection signal and the like. The bioavailability and total amount of cadmium can be analyzed by complementary analysis of the escherichia coli engineered strain and AAS and other physical and chemical analytical methods, and a basis for objective assessment of biological toxicity of cadmium is provided.

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): C12N1/21C12Q1/689C12Q1/02C12R1/19
Inventor 吕建新吕攀攀王伍卢彬彬林炜炜
Owner WENZHOU MEDICAL UNIV
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