Construction Of Nitrophenols Degradation-Tracer Recombinant Plasmid And Ultrasound-mediated Transformation Of Microbial Cells

Nitrophenols are important and commonly used chemical raw materials,which are widely used in synthetic chemicals,pesticides,explosives,dyes,pigments and rubber,They are intermediate metabolite of some organophosphate insecticides such as methyl parathion.Due to the stable structure of the nitrophenol compound,it is difficult to degrade rapidly in the environment.While its wide application causes many environmental problems,microbial-specific degradation is characterized by low-cost and environmental friendliness which can be the main way to remove such pollution.A PNP(p-nitrophenol)degrading strain Methylobacterium sp.C1 was screened from chemical wastewater,and its growth and PNP metabolites were studiedIn this study,the whole genome sequence of C1 strain was determined by combining the second generation sequencing technology(Illumina Miseq sequencing platform)with the third generation sequencing technology(PacBio RS II sequencing platform),and the whole genome sequence of the strain was obtained(NZ_CP 017640).The open reading frame was genetically annotated using KEGG,eggNOG,Swiss-Prot,GO and other databases to find the key monooxygenase(NPM)and dioxygenase(HQD)in the PNP metabolic pathway.The upstream and downstream open reading frames of the single-dual-oxygenase gene were aligned with the protein sequences of known PNP-degrading enzyme-related enzymes,and a sequence of about 27 000 bp found in the whole gene sequence may be a C1 bacterial PNP metabolic gene cluster.The double expression plasmid pCDFDuet-1 was modified in order to allow the uniformoxygenase gene to be constitutively expressed in other strains and to visually observe the distribution and activity of the plasmid and cells.A double expression vector pCDF-GFP constitutively expressing green fluorescent protein was constructed.On this basis,the single-dioxygenase gene was integrated into the pCDF-GFP vector to obtain the recombinant expression vector pCDF-HQNP.In addition,the lac operator and related tag sequences in the pCDF-GFP vector were removed by gene synthesis and restriction enzyme ligation,and finally the recombinant plasmid pCDF-HQNP+with green fluorescent protein tracer and constitutively expressing a single dioxygenase was obtained.Many natural strains cannot be cultured in the laboratory,but traditional cell transformation methods are difficult to implement directly in the environment.Therefore,using the characteristics of in situ transformation by ultrasonic transformation,the role of ultrasound in E.coli transformation is studied.E.coli BL21(DE3)plasmid transformation was carried out in the original culture solution by ultrasonic transformation.The plasmid pCDF-GFP was successfully transferred into the cells in the experiment.Through investigating on the effect of bacterial concentration,treatment time,plasmid concentration and different metal ions,the optimum transformation conditions under different standards were obtained.Under the condition of OD600=1,50 mM CaC12,1 ng/?L plasmid,the highest conversion rate was(1.20±0.06)×106 CFU/ng after sonication for 60 s.