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Escherichia coli for detecting mercury

A technology of Escherichia coli and escherichia coli, which is applied in the field of environmental biology, can solve the problems of high background of plasmid integration, low fluorescence intensity, and weak detection signal, and achieve short detection time, simple equipment and reliable results. letter effect

Inactive Publication Date: 2015-08-12
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Only one fluorescent protein gene gfp is connected in the medium, the fluorescence intensity is not high, and the detection signal is not strong
[0007] The Escherichia coli biological detection strain of the present invention can overcome the shortcomings of high background and instability of the plasmid integration type, construct a specific, sensitive and rapid detection of mercury Escherichia coli strain, for the detection of mercury ions in water lay the foundation

Method used

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  • Escherichia coli for detecting mercury
  • Escherichia coli for detecting mercury
  • Escherichia coli for detecting mercury

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. Fusion of target gene

[0051] 1. Primer information and synthesis

[0052] Synthesize the fusion reporter gene and design the primer sequence according to the searched mercury-regulated gene merR and the promoter PmerT and the cfp gene sequence published in Genebank, as shown in Table 1, P 1 ,P 2 Forward and reverse primers for amplifying merR and promoter PmerT, P 3 ,P 4 Respectively amplify cfp gene forward and reverse primers, P 2 and P 3 The primer has a complementary sequence of about 20bp, and the merR+PmerT and cfp are connected in series by crossover PCR to form a fusion reporter gene. 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, make a storage solution with a concentration of 10 μM / L, and store at -40°C. P 5 ,P 6 and P 7 The primer connects the linker sequence to the cfp gene by PCR, P 9 ,P 10 The primers amplified the li...

Embodiment 2

[0091] Example 2. Gene knock-in

[0092] 1. Synthesis of primer information

[0093] P 11-18 is the gene knock-in primer, where P 11-14 To amplify the N-terminal and C-terminal primers of zntA position respectively, P 11 and P 14 Primers contain Sal I and Not I restriction sites respectively, P 12 and P 13 Containing 20bp homology arms with the common detection element N-terminal and C-terminal respectively, P 15-16 Primers for amplifying the linearized pKOV vector at Sal I and Not I restriction sites, P 17-18 In order to amplify the primers of the mercury detection element, the mercury detection element was connected to the pKOV vector by enzyme-cut ligation, and then the mercury detection element merR::PmerT::cfp::cfp was replaced by the zntA position by gene recombination.

[0094] Table 9

[0095]

[0096] Note: The underlined gene sequence is the restriction site

[0097] 2. Amplification of two homology arms

[0098] Take 1ml of E.coli MC4100 overnight bacte...

Embodiment 3

[0153] Embodiment 3. establish the microbiological method that Escherichia coli bacterial strain E.coli WMC-010 detects mercury ion

[0154] 1 Preparation of culture medium

[0155] 1g / L tryptone, 0.5g / L yeast extract, 1g / L NaCl, first add 50ml MilliQ H 2 O, add MilliQ H after shaking until completely melted 2 O was adjusted to 80ml, 121 ° C, 30min high pressure steam sterilization, then added 10ml 400mM sterile MOPS buffer (pH 7.2), and mixed.

[0156] 2 inoculation

[0157] Take 5-10 μL of the E.coli WMC-010 bacteria solution of the recombinant strain constructed in Example 1 and add it to the 5ml fresh LB liquid medium prepared in step 1, and cultivate overnight at 37°C and 250rpm;

[0158] 3. Establishment of standard curve:

[0159] 3.1 Use a pipette gun to draw the E.coli WMC-010 bacterial solution in step 3.2, and add the calculated E.coli WMC-010 overnight bacterial seed solution to the 50ml fresh LB liquid medium prepared in step 2 to make the initial bacterial so...

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Abstract

The invention provides a construction method of an escherichia coli engineering strain for detecting heavy metal, mercury, and establishment of a method for detecting the mercury in water bodies with the engineering strain, wherein the genetic engineering strain is merR::PmerT::cfp-linker-cfp / E.coli[delta]zntA and is called E.coli WMC-010 (with the accession number of CGMCC No.9749). A met operon sourced from serratia marcescens is knocked-in into a zntA position of a genome of a wild-type E.coli MC4100 strain, thereby starting expression of cyan fluorescent protein (CFP). The engineering strain can overcome the defects that a reported strain containing reported plasmids is high in fluorescent background values, is poor in repeatability of independent experiment and is difficult to accurately and quantitatively detect the mercury in water bodies. The engineering strain can reflect bioavailability of the mercury in the water bodies, thereby providing evidence for objectively evaluating biotoxicity effect of the mercury in the water bodies.

Description

technical field [0001] The invention relates to a microbiological method for detecting mercury, in particular to a method for constructing Escherichia coli modified by genetic engineering technology and a method for establishing the method for detecting mercury in a water body environment, and belongs to the field of environmental biotechnology. Background technique [0002] Mercury, commonly known as mercury, is one of the most toxic heavy metal elements in the environment. It is volatile and liquid at normal temperature and pressure. . The degree of harm of mercury to the human body is related to the chemical form of mercury, among which organic mercury compounds, especially methylmercury, are the most toxic and harmful. A typical mercury pollution incident is the "Minamata disease" incident in Kumamoto Prefecture, Japan. Minamata disease is caused by the fact that mercury is an element that cannot be broken down or degraded into harmless substances. Once released into t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12Q1/68C12Q1/02C12R1/19
CPCC12N1/205C12R2001/19
Inventor 吕建新肖芳兰王伍吴文鹤
Owner WENZHOU MEDICAL UNIV
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