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Whole-cell biosensor for detecting heavy metal ions in water-soluble sample and construction and application thereof

A heavy metal ion, biosensor technology, applied in the field of environmental biology, can solve the problems of not easy to carry instruments, expensive instruments, long detection period and so on

Active Publication Date: 2020-03-10
SOUTH CHINA UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, the detection cycle is long. Although green fluorescent protein has many advantages, it takes a long time to mature, and it usually takes 2 to 3 hours to fully mature, which is not conducive to rapid detection.
Furthermore, the quantification of GFP requires the use of expensive instruments, and the instruments are not easy to carry, which is not conducive to on-site real-time detection

Method used

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  • Whole-cell biosensor for detecting heavy metal ions in water-soluble sample and construction and application thereof
  • Whole-cell biosensor for detecting heavy metal ions in water-soluble sample and construction and application thereof
  • Whole-cell biosensor for detecting heavy metal ions in water-soluble sample and construction and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 Obtainment of heavy metal ions in response to recombinant bacteria

[0085] 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:

[0086] 1. The response proteins and bidirectional promoter sequences (SEQ ID No. 1 and SEQ ID No. 2) responsive to mercury and lead ions were synthesized respectively. The sequences from 5' to 3' were EcoRI, NotI, XbaI, and reverse complement of the response protein Coding gene, bidirectional promoter, SpeI, NotI and PstI.

[0087] 2. Synthetic cleavage gene SRRz, whose sequence is shown in SEQ ID No. 3, the sequence is EcoRI, NotI, XbaI, SRRz cleavage gene, SpeI, NotI and PstI in sequence from 5' to 3'.

[0088] 3. Synthetic terminator T rrnB , its sequence is shown in SEQ ID No.4, the sequence from 5'...

Embodiment 2

[0095] Example 2 Rapid detection of sample heavy metal ion concentration by bacterial cell lysis efficiency

[0096] 1. Recovery and activation of recombinant Escherichia coli

[0097] Recombinant E. coli was streaked into LB plate medium from -80°C refrigerator, and cultured at 37°C for 14 h. A single colony was picked, inoculated into LB medium, and cultured at 37°C and 220rpm for 12-16h.

[0098] 2. Preparation of Escherichia coli detection solution

[0099] Inoculate the revitalized bacterial solution into fresh LB medium at a volume ratio of 1:50, and cultivate it to OD at 35-37 °C and 220 rpm. 600 When the temperature reaches 0.4 to 0.8, 0.1 mM IPTG is added to the conical flask, and the culture is continued for 0.5 h to obtain the Escherichia coli detection solution.

[0100] 3. Contact with the test sample

[0101] The Escherichia coli detection solution in the conical flask was divided into different test tubes, each test tube was 5 mL, 10 μL of the test sample wa...

Embodiment 3

[0116] Example 3 Semi-quantitative and rapid detection of heavy metal ion gel combined with chromogenic gel method

[0117] 1. Preparation of chromogenic gel

[0118] 1.1 Add 0.4 g of agar powder to 20 mL of Z buffer, heat it in a microwave to dissolve, and keep it in a 70°C water bath for later use.

[0119] 1.2 Add 40 mg / mL of X-gal solution to the above solution to a final concentration of 1 mg / mL. After mixing, pipette 200 μL into a 96-well plate, cool at room temperature and solidify and store at 4°C for later use. Can be stored for more than 3 months.

[0120] 2. Drawing of standard curve of color developing gel

[0121] The test bacteria were activated, cultured and contacted with the sample to be tested according to steps 1-3 of Example 2. Add 10 μL of bacterial solution to the well containing the detection gel and incubate at 37°C for 30 min. Different metal ion concentrations, the gel will show different shades of blue ( Figure 4 ).

[0122] Use a camera to ta...

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Abstract

The invention discloses a whole-cell biosensor for detecting heavy metal ions in a water-soluble sample and construction and application thereof. The operation object is escherichia coli, no pathogenic risk exists, and the operation is simple and easy to implement. An SRRz cracking gene is used as a report element, so that microbial bacteria are cracked, and the turbidity of bacterial liquid is obviously changed. Detection can be performed through a visible spectrophotometer, and a result can be obtained through direct observation with naked eyes. The response is quick, and only 30-60 minutesare needed from sample contact to result acquisition. X-gal is used for detecting beta-galactosidase released to the outside of the cells, and the operation is simple and convenient. Expensive instruments are not needed in application, so that the detection cost is greatly reduced, and field detection becomes possible. The extracellular beta-galactosidase activity is accurately quantified by usingchromogenic gel containing oNPG, the detection limit is low, and the detection result is accurate. Technical support is provided for daily heavy metal pollution detection and sudden heavy metal pollution detection.

Description

technical field [0001] The invention belongs to the technical field of environmental biology, and particularly relates to a whole-cell biosensor for highly sensitive detection of heavy metal ions (divalent mercury ions or lead ions) in water-soluble samples, and its construction and application. Background technique [0002] With the development of industry, heavy metal pollution has become increasingly serious and has become one of the prominent environmental problems. Heavy metals are highly toxic, easy to accumulate in organisms and difficult to degrade, and bring great threats to human health after entering the food chain. Therefore, the detection technology of heavy metal ions is particularly important. The detection techniques of heavy metal ions mainly include atomic absorption spectrometry, atomic fluorescence spectrometry, inductively coupled plasma method, ultraviolet-visible spectrophotometry, high performance liquid chromatography, electrochemical analysis, biolo...

Claims

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Application Information

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IPC IPC(8): G01N33/53C12Q1/10C12Q1/34C12R1/19
CPCC12Q1/10C12Q1/34G01N33/53
Inventor 卓敏李爽胡日荣吴柏华彭晓春
Owner SOUTH CHINA UNIV OF TECH
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