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Reporting bacterial strain sensitive to oxidation-reduction cycle reactant and preparation method thereof

A cycle reaction and bacterial strain technology, applied in biochemical equipment and methods, methods based on microorganisms, bacteria, etc., can solve the problems of large subjective errors, low accuracy, time-consuming and labor-consuming oxidation-reduction cycle reagents, etc., to achieve Easy to construct carrier, high sensitivity effect

Inactive Publication Date: 2009-03-18
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The object of the present invention is to construct a kind of stable Escherichia coli reporter strain as the reaction of the biosensor of detection redox cycle reactant by gene knockout and gene replacement technology Host, to quickly and sensitively detect redox cycle reactants to solve the shortcomings of growth curve method, endpoint method, and MIC method to detect redox cycle reactants that are time-consuming, labor-intensive, subjective error is large, and accuracy is not high

Method used

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  • Reporting bacterial strain sensitive to oxidation-reduction cycle reactant and preparation method thereof
  • Reporting bacterial strain sensitive to oxidation-reduction cycle reactant and preparation method thereof
  • Reporting bacterial strain sensitive to oxidation-reduction cycle reactant and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1. Fusion of target gene

[0056] (1) Primer information and synthesis

[0057] According to the gene knockout primer sequences provided by the Harvard University website and the SoxS and GFPmut2 gene sequences published by GenBank, the inner primers (P2:soxS-Ni and P3:soxS-Ci) and outer primers (P1 :soxS-No and P4:soxS-Co), so that there is a complementary sequence between P2 and P5, P3 and P6, the two outer primers for gene knockout remain unchanged, and P5 and P6 are a pair of primers for amplifying the GFPmut2 gene. The specific information is shown in Table-1. The primers were synthesized by Dalian Bao Biology Co., Ltd. The primers were dissolved in sterile deionized water to make a concentration of 10 μmol / L.

[0058] Table 1

[0059]

[0060] (2) Cross PCR for gene fusion

[0061] Using cross-PCR technology to carry out gene fusion through three-step PCR, the two-terminal sequence of the target gene SoxS is fused with the GFPmut2 gene to form a mod...

Embodiment 2

[0062] Example 2. Amplification of two homology arms and the GFPmut2 gene

[0063] 1. Amplification of two homology arms:

[0064] Use an inoculation needle to pick a small amount of a single colony of E.coli MC4100 on the LB plate, mix it in a 200 μL PCR reaction tube containing 15 μL sterile water, cook in boiling water for 10 minutes, and centrifuge at 12 000 rpm for 3 to 5 minutes to remove the water droplets condensed on the lid from the tube. end. Add 5 μL of the system prepared in Table 2 into the tube and mix well.

[0065] Table 2

[0066]

[0067] The PCR reaction conditions were as follows: 1 min at 94°C; 4 min at 88°C; 10 sec at 94°C, 3 min at 66°C, 25 cycles; 10 min at 72°C, and store at 4°C. 5 μL of the product was identified by 1.0% Agarose gel electrophoresis. The PCR product was purified with a PCR product purification kit, and finally dissolved and eluted by adding an appropriate amount of ddH2O for later use.

[0068] 2. Amplification of GFPmut2 gene: ...

Embodiment 3

[0080] Embodiment 3, PCR product adds A reaction

[0081] Add Ex Taq to the above PCR reaction product at a ratio of 5 U per 50 μL Polymerase, 72°C 10min.

[0082] (4), PCR fragment purification and recovery

[0083] Add A reaction product to purify with a PCR product purification kit, and finally add an appropriate amount of ddH2O to dissolve and elute for later use.

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Abstract

The invention relates to an Escherichia coli report bacterial strain and a preparation method thereof, in particular to a report bacterial strain with high sensitivity on an oxidation reduction cycle reactant and a preparation method thereof. The Escherichia coli report bacterial strain is prepared by deleting five genes, namely soda, sodB, katG, ahpCF and frdABCD, of wild Escherichia coli E.coli MC4100 and replacing an SoxS encoding gene with GFP by the gene substitution technology. The Escherichia coli report bacterial strain is named as E.coli WMC-002, and is preserved in the China General Microbiological Culture Collection Center, with a preserving number of CGMCC No.2464. The Escherichia coli report strain can solve the problem of the limitation of detection of the oxidation reduction cycle reactant by the growth curve method, the end-point method and the MIC metho, simultaneously can overcome the defects of high background of a report signal of the report bacterial strain provided with a report vector, relative difficulty in precise quantification, poor stability and so on, and can be used for biological detection of the oxidation reduction cycle reactant.

Description

technical field [0001] The present invention relates to a bacterial strain transformed by metabolic engineering by means of molecular biology, in particular to an Escherichia coli reporter strain highly sensitive to redox cycle reactants, a preparation method and its application in detecting redox cycle reactants application. Background technique [0002] In recent years, with the rapid development of industry and the improvement of people's living conditions, people are paying more and more attention to environmental issues. "Environmental protection, ecology, green, health" has become the theme concept of people's life in the 21st century. [0003] Redox cycling reagents are a class of serious industrial and agricultural pollutants. These substances have the characteristics of enzymes, which enable cells to continuously produce superoxide anions, and at the same time cause a large amount of reducing equivalents (NADH / NADPH) in cells. Consumption, the produced superoxide ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/09C12N15/63C12Q1/02C12R1/19
Inventor 吕建新王茂峰童贞珍
Owner WENZHOU MEDICAL UNIV
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