Genetically engineered bacterium for rapidly degrading methyltestosterone and application thereof
A technology of methyl testosterone and genetically engineered bacteria is applied in the field of application of environmental microorganisms to achieve the effects of improving degradability, strong adaptability and high removal efficiency
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Embodiment 1
[0029] According to the gene sequence (GenBank accession No. AHIL00000000), the gene map was drawn centering on the 3,17β-hydroxysteroid dehydrogenase gene, and other genes close to the 3,17β-hydroxysteroid dehydrogenase gene were selected to analyze their expression and production The properties of the protein, the final selection PhaR gene is the target gene, the result is as follows figure 1 shown.
Embodiment 2
[0031]Total DNA was extracted from Comamonas testosteroni grown overnight in LB medium by CTAB method. According to the gene sequence in GenBank, an additional base G was added behind the third base at the 5' end of the PhaR gene to design the forward primer, that is, the forward primer was 5'-ATGGCAAGAAAACAAAGAG-3', and the reverse primer was 5' '-CTGCATGGCATGACCATA-3'. Using the total DNA as a template, carry out PCR amplification and use PCR technology to obtain the first 300 bp mutant gene fragment of the PhaR gene from the genome of Comamonas testosteroni (such as figure 2 -a), and then clone the mutated gene fragment into the expression vector pCR2.1-TOPO to obtain the expression vector pPK-8 containing the mutated gene fragment, and prove that the plasmid pPK-8 is the target plasmid by plasmid size and EcoRI fragment (like figure 2 -b), and then use the electroporation introduction method (pulse voltage 1.8kv, time 4.9 milliseconds) to integrate the pPK-8 plasmid in...
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