Semi-quantitative detection method for heavy metal ions in water-soluble sample based on whole-cell biosensor

A heavy metal ion and biosensor technology, applied in the field of environmental biology, can solve the problems of long response time of fluorescent protein expression, high background signal, and practical obstacles, and achieve easy high-throughput screening, good safety, and easy operation Effect

Pending Publication Date: 2022-05-27
SOUTH CHINA UNIV OF TECH
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  • Claims
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AI Technical Summary

Problems solved by technology

Green fluorescent protein (GFP) from jellyfish produces fluorescence without adding any exogenous matrix, but due to the long response time and high background signal of fluorescent protein expression, it is necessary to use a UV spectrophotometer to determine the concentration, which is expensive and cumbersome to operate , whose utility has been hampered in bacterial biosensors

Method used

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  • Semi-quantitative detection method for heavy metal ions in water-soluble sample based on whole-cell biosensor
  • Semi-quantitative detection method for heavy metal ions in water-soluble sample based on whole-cell biosensor
  • Semi-quantitative detection method for heavy metal ions in water-soluble sample based on whole-cell biosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 Obtainment of heavy metal ions in response to recombinant bacteria

[0082] Using pSB1C3 as the starting vector (iGEM, http: / / parts.igem.org / Part:pSB1C3), a heavy metal response vector was constructed. The schematic diagram of the construction of the heavy metal response vector is as follows figure 1 shown. The specific construction method is as follows:

[0083]1. Synthesize the DNA sequence of CadA operon that specifically recognizes Cd(II), including Cd(II) response protein and bidirectional promoter (SEQ ID No. 1), the sequence is EcoRI, NotI, XbaI from 5' to 3' , Response protein reverse complement coding gene, bidirectional promoter, SpeI, NotI and PstI.

[0084] 2. Synthetic cleavage gene SRRz, whose sequence is shown in SEQ ID No. 2, the sequence is EcoRI, NotI, XbaI, SRRz cleavage gene, SpeI, NotI and PstI in sequence from 5' to 3'.

[0085] 3. Synthesize the terminator TrrnB, the sequence of which is shown in SEQ ID No. 3. The sequence is EcoRI, ...

Embodiment 2

[0090] Example 2 Rapid detection of sample heavy metal ion concentration by bacterial cell lysis efficiency

[0091] 1. Recovery and activation of recombinant Escherichia coli

[0092] Recombinant E. coli was streaked in LB plate medium from -80°C refrigerator, and cultured at 37°C for 15h. A single colony of the Cd(II) detection gene biosensor E.coli BL21-MCd was picked from the plate, and then inoculated into 5 mL of liquid LB medium containing chloramphenicol with a final concentration of 25 μg / L at 37°C, 220rpm, overnight culture for more than 15h.

[0093] 2. Preparation of Escherichia coli detection solution

[0094] The resuscitated and activated bacterial solution was transferred to fresh LB liquid medium at 1:50 (volume ratio), and IPTG inducer was added to make the final concentration 0.01 mmol / L. Shaker culture (37°C, 220rpm) to OD 600 The absorbance value was about 0.6, and the detection solution of Escherichia coli was obtained.

[0095] 3. Contact with the t...

Embodiment 3

[0108] Example 3 Semi-quantitative rapid detection of heavy metal ions in combination with chromogenic method

[0109] 1. Preparation of color system

[0110] Add 40mg / mL X-gal solution to Z buffer to a final concentration of 1mg / mL, mix well and add to 96-well plate for later use.

[0111] 2. Color rendering standard curve drawing

[0112] The test bacteria were activated, cultured and contacted with the sample to be tested according to steps 1 to 3 of Example 2.

[0113] The bacterial liquid in the 96-well plate was transferred to a centrifuge tube, centrifuged at 8000 g for 3 min, and the supernatant was taken. Prepare a β-galactosidase catalytic color detection system according to Table 1.

[0114] Table 1 Preparation of color detection system

[0115] reagent Volume (μL) X-gal (40mg / mL) 6 Bacterial liquid supernatant 30 Z buffer 204

[0116] Among them, Z buffer preparation method: add 0.12g MgSO to 50mL 1×PBS buffer solution 4 and ...

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Abstract

The invention discloses a semi-quantitative detection method for heavy metal ions in a water-soluble sample based on a whole-cell biosensor, and belongs to the technical field of environmental biology. The method is convenient to operate, short in time consumption, low in cost, good in visibility and easy to quantify. The SRRz cracking gene is used as a report element, the response is quick, and the optical density of the bacterial liquid is obviously reduced within 2 hours when the bacterial liquid is in contact with heavy metal. According to the present invention, the SRRz splitting gene is adopted as the report element so as to cause the splitting of the microbial thalli, the correlation between the obtained result and the Cd (II) ion concentration is high, and the detection result can be determined by using naked eyes, and can be detected through a visible light spectrophotometer; x-gal is used for detecting the beta-galactosidase released outside the cells, and the operation is simple and convenient. The method is good in safety, free of reagent addition, free of pigment interference, short in time consumption, low in cost, specific, easy to realize high-throughput screening and convenient to popularize; and technical support can be provided for daily detection and high-throughput screening of heavy metal pollution.

Description

technical field [0001] The invention belongs to the technical field of environmental biology, and particularly relates to a semi-quantitative detection method for heavy metal ions (divalent cadmium ions) in water-soluble samples based on a whole-cell biosensor. Background technique [0002] With the continuous enhancement of people's awareness of environmental protection, more and more attention is paid to water pollution, especially the pollution of water bodies by heavy metal elements. They are not biodegradable, but have a tendency to accumulate in living organisms, causing chronic poisoning. Therefore, its determination is of great significance for both human health and environmental protection. There are two main development directions for the detection of heavy metal ions. One is instrument-based analytical chemistry methods, such as gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), atomic absorption spe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12N15/65C12N15/33C12Q1/10C12Q1/34C12R1/19
CPCC12N15/70C12N15/65C07K14/005C12Q1/10C12Q1/34C12N2830/205C12N2795/10322Y02A50/30
Inventor 李爽马浩朱晁谊
Owner SOUTH CHINA UNIV OF TECH
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