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Recombinant strain representing genetic toxicity, construction method and application thereof

A recombinant bacteria and the technology in the sequence listing, which is applied to the recombinant bacteria for characterization of genotoxicity and its construction and application fields, can solve the problems of inconstant copy number and unstable expression of biosensing cells, etc.

Active Publication Date: 2009-09-23
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 strain representing genetic toxicity, construction method and application thereof
  • Recombinant strain representing genetic toxicity, construction method and application thereof
  • Recombinant strain representing genetic toxicity, construction method and application thereof

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 strain representing genetic toxicity, a construction method and application thereof. The recA gene of the chromosome in the recombinant strain includes the Acinetobacter (Acinetobacter sp.) ADP1 of a reporter gene. The reporter gene has the same promoter with the recA gene. The recA gene therein further contains a selective marker gene that is located at the terminal 3' of the reporter gene. The selective marker gene carries a promoter. The recombinant strain is exposed in substances with genetic toxicity or radiation with DNA damage capacity, the reporter gene expresses and generates reporter products, the amount of the reporter products has a dose-effect relationship with the genetic toxicity. The recombinant strain can be used for evaluating the genetic toxicity of a sample and the DNA damage intensity of radiation.

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 Applications(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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