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Recombinant bacteria for characterizing genotoxicity and its construction method and application

A genotoxic, recombinant bacteria technology, applied in the direction of microorganism-based methods, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of unstable expression of biosensing cells, and the number of copies is not fixed.

Active Publication Date: 2012-02-22
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the copy number of the plasmid is not fixed, which will cause unstable expression of biosensor cells

Method used

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  • Recombinant bacteria for characterizing genotoxicity and its construction method and application
  • Recombinant bacteria for characterizing genotoxicity and its construction method and application
  • Recombinant bacteria for characterizing genotoxicity and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1. Construction of Acinetobacter sp.ADP_recA_km_lux

[0032] 1) Construction of BamHI and EcoRI restriction endonuclease sites in the recA gene of Acinetobacter sp. ADP1

[0033] Acinetobacter sp.ADP1 chromosomal DNA has been fully sequenced and is available from the NCBI Genebank database.

[0034] The following primers were designed to obtain the 5' part and the 3' part of the complete recA gene of Acinetobacter ADP1 by PCR reaction:

[0035] P1: 5'-TCATTAGCAATAAGAGGTTGGATCG-3'

[0036] P2: 5'-TCGCACCAGAGCGTACAAGCGGATCCGGAATTCCC-3'

[0037] P3: 5'-GGGAATTCCGGATCCGCTTGTACGCTCTGGTGCGA-3'

[0038] P4: 5'-GGCTTCGCAATTGTACTCTGTG-3'

[0039] Wherein, P1 and P2 are a pair of primers for amplifying the 5' part of the recA gene; P3 and P4 are a pair of primers for amplifying the 3' part of the recA gene.

[0040] Enzyme recognition sites for EcoRI and BamHI were designed on primers P2 and P3, respectively.

[0041] Using Acinetobacter ADP1 cells (Huang, W.E., ...

Embodiment 2

[0058] Example 2. The response of Acinetobacter sp.ADP_recA_km_lux to the genotoxic substance mitomycin C

[0059] 1) Cell culture and preparation

[0060] A single colony of Acinetobacter sp.ADP_recA_km_lux was inoculated into LB medium (LBK) containing 10 mg / L kanamycin, and cultured overnight at 30°C. Dilute the cells 10-25 times with fresh LBK medium to obtain a cell suspension for use.

[0061] 2) Sample preparation

[0062] Aqueous solutions of mitomycin C with concentrations of 0.0001, 0.001, 0.01, 0.1 and 1 ug / ml were prepared respectively.

[0063] 3) Sample test

[0064] Take 200 μL of the cell suspension diluted in step 1), and add it into the wells of the black-bottomed 96-well enzyme plate. 2 μL of mitomycin C aqueous solution (0.03, 0.3 and 3 μM) of different concentrations were added to each well, and clear water was used as a negative control. A multi-function microplate reader (SpectraMax M2 plate reader, Molecular Devices Corporation) capable of measurin...

Embodiment 3A

[0068]Example 3 Detection of the genotoxicity of polycyclic aromatic hydrocarbons by Acinetobacter sp.ADP_recA_km_lux

[0069] 1) Cell culture and preparation

[0070] A single colony of Acinetobacter sp.ADP_recA_km_lux was inoculated into LB medium (LBK) containing 10 mg / L kanamycin, and cultured overnight at 30°C. Dilute the cells 10-25 times with fresh LBK medium to obtain a cell suspension for use.

[0071] 2) Sample preparation

[0072] Dissolve pyrene in DMSO solvent, and prepare pyrene solutions with concentrations of 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1 and 1 mM.

[0073] 3) Sample test

[0074] Dissolve 2 μL of pyrene solution in 18 μL of high-purity water, and add 2 μL of S9 liver mitochondrial enzyme (Sigma, S2442) and 3 μL of high-purity water. Then mix with 180 μL of the cell suspension diluted in step 1) in a 96-well plate. A multi-function microplate reader (SpectraMax M2 plate reader, Molecular Devices Corporation) capable of measuring chemiluminesc...

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Abstract

The invention discloses a recombinant bacterium characterizing genotoxicity, its construction method and application. The recombinant bacterium is Acinetobacter sp. ADP1 containing a reporter gene in the recA gene of the chromosome; the reporter gene and the recA gene have a common promoter. The recA gene may also contain a screening marker gene, which is located at the 3' end of the reporter gene; the screening marker gene has a promoter. After the recombinant bacteria are exposed to genotoxic substances or radiation with DNA damage ability, the reporter gene expression produces a reporter product, and the amount of the reporter product has a dose-effect relationship with the genotoxicity, which can be used to evaluate the genotoxicity of the sample and the DNA of the radiation damage intensity.

Description

technical field [0001] The invention relates to a recombinant bacterium for characterizing genotoxicity and its construction method and application. Background technique [0002] With the development of modern industry, environmental pollution is becoming more and more serious, and human beings discharge a large amount of toxic and harmful substances into the environment, endangering human health, so the detection of toxic substances is very important. Among them, the emphasis on genotoxic substances and the detection of genotoxicity have been the areas that have attracted much attention in the past ten years. There are usually two methods for the detection of genotoxic substances - physical and chemical methods and biological methods. Physicochemical methods are based on instrumental analysis of sample components, such as GC-MS or HPLC. The composition data of the sample can be obtained by physical and chemical means, and the toxicity of the substance in the sample can be...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12Q1/02C12R1/01
Inventor 李广贺黄巍宋一之
Owner TSINGHUA UNIV
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