A recombinant bacterium characterizing genotoxicity and its construction method and application
A genotoxic, recombinant bacteria technology, applied in microorganism-based methods, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problem of inability to express the repressor protein LexA degradation or binding, no response to genotoxic substances, inability to repeat, etc. question
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Embodiment 1
[0064] Construction of HesenGTox3
[0065] 1) Obtaining the natrigovibrio ( Vibrio natriegens )of recA gene promoter
[0066] Vibrio natriegens Purchased from ATCC under Accession No. 14048.
[0067] Vibrio natriegens Chromosomal DNA has been fully sequenced and is available from the NCBI Genebank database.
[0068] Design the following primers to obtain by PCR reaction Vibrio natriegens complete recA Gene promoter:
[0069] P1: 5’-CATGGCCGCGGGATTCGTGATAAGCTCTGCGGCA-3’
[0070] P2: 5’-CTCCAAGCTTACTTTCTCCGATTTATTCATCTTTTTGGGCGAC-3’
[0071] by Vibrio natriegens The genome is used as a template, and the reaction conditions for PCR are as follows:
[0072]
[0073] After the PCR product was subjected to 1% agarose electrophoresis, it was obtained by gel cutting and purification using Qiagen’s QIAquick gel extraction kit recA The promoter part of the gene.
[0074] 2) luxCDABE Gene acquisition
[0075]Using pSalAR_lux as a template, the plasmid has been d...
Embodiment 2
[0102] Hesen GTox3 Response to the genotoxic substance mitomycin C
[0103] 1) Cell culture and preparation
[0104] Hesen GTox3 A single colony was inoculated into LB medium (LBA) containing 100 μg / mL ampicillin, and cultured overnight at 37°C. Cells were collected at 4°C at 3000 rpm for 5 min, washed 3 times with PBS, then diluted 10 times with M9 medium and resuspended to obtain a cell suspension for use.
[0105] 2) Sample preparation
[0106] Mitomycin C (MMC) aqueous solutions were prepared at concentrations of 0, 150 nM, 1500 nM, 15000 nM, 150 µM and 1500 µM, respectively.
[0107] 3) Sample test
[0108] Take 200 μL of the cell suspension diluted in step 1), and add it into the wells of the black-bottomed 96-well ELISA plate. Add 2 μL of mitomycin C aqueous solution of different concentrations to each well, and clean water was used as a negative control. A multifunctional microplate reader (SpectraMax M2 plate reader, Molecular Devices Corporation) that can me...
Embodiment 3
[0113] Hesen GTox3 Detection of UV genotoxicity
[0114] 1) Cell culture and preparation
[0115] Hesen GTox3 A single colony was inoculated into LB medium (LBA) containing 100 μg / mL ampicillin, and cultured overnight at 37°C. Cells were collected at 4°C at 3000 rpm for 5 min, washed 3 times with PBS, then diluted 10 times with M9 medium and resuspended to obtain a cell suspension for use.
[0116] 2) Sample preparation
[0117] Take 200 μL of the cell suspension diluted in step 1), and add it into the wells of a black bottom-transparent 96-well microtiter plate. Then put the ELISA plate into a UV crosslinker (UVP, CL-1000), 8 W, 300 s.
[0118] 3) Sample test
[0119] After exposure for 300 s, the luminescence intensity of UV-induced recombinant bacteria was photographed in a dark box.
[0120] Hesen GTox3 Response to UV as Figure 5 shown.
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