Recombinant luminous bacteria and application thereof

A technology of recombining bacteria and bacteria liquid, applied in bacteria, chemiluminescence/bioluminescence, analysis by chemical reaction of materials, etc., can solve the change of luminescence expression of natural luminescent bacteria, difficult to evaluate the actual effect of mercury, change heavy metal Bioavailability and other issues, to achieve the effect of easy automatic online, high reliability of results, and avoidance of false negative problems

Active Publication Date: 2014-11-12
TSINGHUA UNIV
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  • Claims
  • Application Information

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Problems solved by technology

Although these physical and chemical detection methods can achieve high-precision and high-sensitivity detection of mercury content in water, it is difficult to evaluate the actual impact of mercury on organisms
Although the luminescent bacteria detection methods in GB/T15441-1995 and ISO11348-2007 can also be used for the biological toxicity evaluation of mercury, the natural l

Method used

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  • Recombinant luminous bacteria and application thereof
  • Recombinant luminous bacteria and application thereof
  • Recombinant luminous bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1, Construction of mer-mercury-responsive recombinant luminescent bacteria

[0047] When constructing mer recombinant luminescent bacteria, the regulatory recognition element composed of R gene and o / p site is inserted into the upstream of the luminescent gene lacking the promoter element, and introduced into suitable recipient cells to obtain mer recombinant luminescent bacteria that can specifically recognize mercury .

[0048] Using the genomic DNA of Pseudomonas putida strain SP1 as a template, design the upstream primer 5'-CGC GGATCC GCATTTCTCCTTTCGA-3' (SEQ ID No.1, the sequence shown by the underline is BamH Ⅰ enzyme digestion recognition site) and the downstream primer 5'-CCGGAATTCTTACGCAACGGGAAAT-3' (SEQ ID No.2, the sequence shown by the underline is EcoR Ⅰ enzyme digestion Recognition site) for PCR amplification to obtain the merRo / p sequence in genomic DNA, the sequence is shown in SEQ ID No.3, the 10th to 80th positions from the 5' end in SEQ ID N...

Embodiment 2

[0051] Example 2, detection of mercury in water samples with recombinant luminescent bacteria mer-pUCD615

[0052] The recombinant luminescent bacterium mer-pUCD615 can achieve a single specific response to heavy metal mercury without interference from other metal pollutants such as cadmium and lead, and there is no false positive problem. Under the condition of low cytotoxicity, as long as there is bioavailable mercury in the water sample, the strain can change from non-luminescent to continuous luminescent, and then detect the luminous intensity by modern photon detection technology, and use this strain to determine the bioavailable mercury in the water body. The specific steps for the bioavailable mercury concentration range are as follows:

[0053] 1. Preparation of bacteria solution for testing

[0054] Both methods shown in (1) or (2) below are acceptable.

[0055] (1) Inoculate the recombinant luminescent bacterium mer-pUCD615 in LB medium and culture it in a shaker a...

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Abstract

The invention discloses recombinant luminous bacteria and application of the recombinant luminescent bacteria. According to the recombinant luminescent bacteria, the R genes in mer genes and a regulation and control recognition element composed of o/p loci are inserted in the upstream of luminous genes lacking a starting element on a carrier and led into recipient bacteria so that the recombinant luminous bacteria can be obtained. The recombinant luminous bacteria with mercury specific response serve as testing strains, and the bioavailability range of mercury concentration in water can be judged according to luminous intensity. Compared with a traditional physicochemical detection method, the method is low in cost, easy to operate, high in result reliability and capable of achieving automatic online detection easily without special high-precision analytical instruments and detecting devices, the method can be applied to the field of environmental water quality monitoring and has great economical and beneficial significance in early warning and monitoring of mercury pollutants.

Description

technical field [0001] The invention relates to a recombinant luminescent bacterium and its application, in particular to a recombinant luminescent bacterium and its application in detecting mercury content, belonging to the field of biotechnology. Background technique [0002] Mercury pollution is produced by metal smelting, manufacturing of scientific measuring instruments, and burning of fossil fuels. Similar to other heavy metals, mercury pollution is persistent, and in micro-dose exposures, through bioaccumulation and biomagnification, making mercury-containing pollutants have a large impact on any ecosystem. Among them, mercury, which is a bioavailable component, contributes the most to pollution. [0003] At present, the standardized mercury measurement methods in my country are mainly spectroscopic methods, such as atomic absorption spectrometry, etc., while the on-line monitoring methods are mainly electrochemical methods based on anodic stripping voltammetry. Alt...

Claims

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Application Information

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IPC IPC(8): C12N1/21G01N21/76C12R1/19
Inventor 汪用志何苗
Owner TSINGHUA UNIV
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