Genetically engineered bacterium for characterizing genetic toxicity as well as preparation and application of genetically engineered bacterium
A technology of genetically engineered bacteria and genotoxicity, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, bacteria, etc., can solve the problems of unstable copy number and unstable expression of biosensing cells
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Embodiment 1
[0034] Embodiment 1. Construction of genetically engineered bacteria Acinetobacter sp.Genotox1
[0035] 1) Create BamHI and EcoRI restriction endonuclease sites in the lexA gene of ADP1.
[0036] Acinetobacter sp.ADP1 chromosomal DNA has been fully sequenced and is available from the NCBI Genebank database. Therefore, the following primers are designed to obtain the upper half and the lower half of the complete lexA gene of Acinetobacter ADP1 by PCR reaction:
[0037] P1:5'-ATGTTGGCTATCGAAGGATCG-3'
[0038] P2: 5'-GATTCCGTACAAGCGGCACCAATTCCCAGAGCCGG-3'
[0039] P3:5'-ATTCAGATCCGCTTGTCTGGTGCGAACGCT-3'
[0040] P4:5'-ATTGTGGCTTCGCATGTGACTC-3'
[0041] Wherein P1 and P2 are a pair of primers for amplifying the upper half of the lexA gene; P3 and P4 are a pair of primers for amplifying the lower half of the lexA gene. Restriction sites for EcoRI and BamHI were designed on primers P2 and P3, respectively.
[0042] Using Acinetobacter ADP1 cells as a template, the specific rea...
Embodiment 2
[0060] Example 2. The response of Acinetobacter sp.Genotox1 to the genotoxic substance mitomycin C
[0061] 1) Cell culture and preparation
[0062] A single colony was picked from the plate, inoculated into LB medium (LBK) containing 10 mg / L kanamycin, and cultured overnight at 30°C. Dilute the cells 10-25 times to fresh LBK medium before the test and set aside.
[0063] 2) Sample preparation
[0064] Prepare an aqueous solution of mitomycin C (MMC).
[0065] 3) Sample test
[0066] Take 180 μL of the bacterial solution diluted in step 1) and add it to the wells of the black-bottomed 96-well enzyme plate. Add 20 μL predicted sample MMC or clear water to each well as a negative control. A multi-function microplate reader (SpectraMax M2 plate reader, Molecular Devices Corporation) capable of measuring chemiluminescence and absorbance in 96-well plates was used. The chemiluminescence (lum) of each microwell was measured at 37°C, and the absorbance of the well at a waveleng...
Embodiment 3
[0070] Example 3 Detection of the toxicity of Acinetobacter sp.Genotox1 to environmental samples
[0071] 1) Cell culture and preparation
[0072] A single colony was picked from the plate, inoculated into LB medium (LBK) containing 10 mg / L kanamycin, and cultured overnight at 30°C. Dilute the cells 10-25 times to fresh LBK medium before the test and set aside.
[0073] 2) Sample preparation
[0074] The ambient samples were left at room temperature for 20 minutes.
[0075] 3) Sample test
[0076] Take 20 μL of the environmental sample, and then mix it with 180 μL of the bacterial solution diluted in step 1 in a 96-well plate. A multi-function microplate reader (SpectraMax M2 plate reader, Molecular Devices Corporation) capable of measuring chemiluminescence and absorbance in 96-well plates was used. The chemiluminescence (lum) of each microwell was measured at 37°C, and the absorbance of the well at a wavelength of 600 nm (OD600) was read. Measurements were taken every ...
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