A strain of Escherichia coli that detects copper

A technology of Escherichia coli and escherichiacoli, applied in the field of environmental biology, can solve the problems of poor signal stability of fluorescence detection and poor repeatability of independent experiments, slow development of heavy metal microorganism detection technology, and uneven copy number of plasmids. The effect of short time, small deviation of fluorescence intensity value, and improved sensitivity

Active Publication Date: 2019-01-25
WENZHOU MEDICAL UNIV
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  • Abstract
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Problems solved by technology

[0009] After retrieval, most of the reporter genes of microbial reporter strains / whole-cell microbial sensors for the detection of heavy metal copper in domestic and foreign literature are connected to plasmid vectors, so there will be high fluorescence background values ​​of reporter genes and the offspring of bacteria after passage The number of plasmid copies in cells is not uniform (too high or missing), which leads to defects such as fluorescence detection signal stability and poor repeatability of independent experiments
In addition, when the heavy metal content in the cells of the wild-type strain is high, the bacteria will start to express the "efflux pump" gene to pump out the self-protection mechanism of heavy metals, resulting in low detection sensitivity and high concentration in the linear range, and the minimum detection limit is higher than National standard limits for copper in water bodies
Due to the above reasons, the application of heavy metal microbial detection technology is limited and the development is slow

Method used

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  • A strain of Escherichia coli that detects copper
  • A strain of Escherichia coli that detects copper
  • A strain of Escherichia coli that detects copper

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1. Detect the preparation of heavy metal copper Escherichia coli strain:

[0065] 1. Construction of Escherichia coli mutant strain E.coli copA sensitive to copper ion by using Red recombination system - cueO - cusA -

[0066] Using pKD3 as a template, the chloramphenicol resistance gene with FRT site (FLP recombinase recognition site) was amplified by PCR, and it was electrotransformed into MC4100 electroporation-competent cells containing pKD46 plasmid. pKD46 / MC4100 bacteria were induced by 30mmol / L arabinose during the competent preparation, so that the pKD46 plasmid expressed Gam, Bet and Exo three lambda phage recombinases in the bacteria, and the Gam recombinase inhibited the RecBCD exonuclease of Escherichia coli Enzyme V activity, so that the chloramphenicol gene electrotransferred into the bacteria will not be degraded immediately. At the same time, Exo and Bet recombinase guide the recombination and replacement of the chloramphenicol gene and the...

Embodiment 2

[0083] Example 2. Determination and parameter optimization of the performance of detecting heavy metal copper Escherichia coli strains

[0084] Optimize the measurement conditions and determine the relevant parameters for the constructed Escherichia coli strains to determine a set of stable, sensitive and accurate experimental measurement conditions, including induction kinetic experiments, determination of response curves, specificity and selectivity experiments , characterization analysis, determination of the optimum detection range, determination of the lowest detection limit, etc.

[0085] 1. Optimization of incubation time of Escherichia coli strain E.coli WMC-007 with copper ions

[0086] 1.1 On fixing Cu 2+ Kinetic analysis of induction of GFPmut2 expression by E. coli WMC-007 under concentration conditions.

[0087] Use a pipette gun to draw a certain volume of E.coli WMC-007 overnight bacterial seed solution and add it to 50ml of fresh LB liquid medium, so that the...

Embodiment 3

[0126] Example 3. Establishment of a methodology for the detection of heavy metal copper in water by Escherichia coli strain E.coli WMC-007 and its application implementation

[0127] 1. Preparation of medium: 1g / L tryptone, 0.5g / L yeast extract, 1g / L NaCl, first add 50ml MilliQH 2 O, add MilliQ H after shaking until completely melted 2 O was adjusted to 80ml, autoclaved (121°C, 30min), then added 10ml of 400mM sterile MOPS buffer (pH 7.2), and mixed.

[0128] 2. Establishment of standard curve:

[0129] Use a pipette gun to draw a certain volume of E.coli WMC-007 overnight bacterial seed solution and add it to 50ml of fresh LB liquid medium (containing MOPS with a final concentration of 40mmol / L), so that the OD600 value of the initial bacterial solution is about 0.02, Aliquot (1ml / tube), add an equal volume of Cu with different concentrations 2+ Solution to each tube of bacteria solution, so that the final concentration is 0.16, 0.39, 0.78, 1.56, 7.81, 15.63, 31.25, 39.06...

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Abstract

The invention provides a preparation method for an engineering strain of Escherichia coli used for detecting the heavy metal copper and establishment of a method for detecting copper in a water body by using the engineering strain of Escherichia coli. The engineering strain provided by the invention has the technical characteristics of specificity, sensitivity, rapidness, cheap price, stability and capacity of quantitative determination of the concentration and bio-availability of copper in a water body. The engineering strain has a wide detection linear range and high sensitivity in detection of copper ions, wherein the detection linear range is 0.39 to 94 [mu]M and lowest detection limit is 0.25 [mu]M; the engineering strain is applicable to both quantitative determination of copper in water bodies like various sewage, domestic drinking water, acidic mine wastewater and electroplating wastewater and to assessment of biotoxicity of treated or restored copper-contaminated water bodies; so the engineering strain and physical and chemical analytical methods like atomic absorption spectrometry can realize complementary analysis of bio-availability and total amount of copper in water bodies, thereby providing bases for objective evaluation of the biotoxicity of the heavy metal copper.

Description

technical field [0001] The invention relates to a construction method for detecting heavy metal copper Escherichia coli engineering strains and a specific implementation scheme for detecting heavy metal copper in water, belonging to the field of environmental biotechnology. Background technique [0002] Copper (Cu), as one of the most common heavy metal elements, is an essential trace element for the metabolism of organisms. Studies have found that excessive copper or copper ions are toxic to all living organisms. Excessive deposition of copper in the human body is involved in the occurrence and development of many diseases. Copper can accumulate in vital organs of the human body, such as liver, kidney, brain, etc. Especially certain people with chromosomal recessive disorders. Due to the dysfunction of bile excretion of copper, copper accumulates in the liver, causing liver damage, chronic and active hepatitis symptoms, such as cirrhosis in Indian children, chronic interst...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C12Q1/6897C12Q1/02C12R1/19
Inventor 吕建新庞一林谭国强刘佳明
Owner WENZHOU MEDICAL UNIV
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