Construction Of Cold Inducible Expression Vector PSW4 And Expression Of DNA Polymerase Iii Epsilon Subunit

The deep-sea derived shutter vector pSW2 has been constructed previously.In this study,a cold inducible expression vector was constructed based on pSW2,thus provide useful explants material and toolbox for construction of engineered strain which can degradate environmental pollutants.The mutation experiments were performed to identify the essential gene in the vector,and GFP was used as reporter gene to testify the expression efficiency of the mutated vectors.Finally,the cold inducible vector pSW4 was constructed by further adding purification tag and multiple cloning site and replacement of the promoter region.The mutation experiment indicated that the GFP intensity was significantly increased by deletion of fpsB gene,which encodes the ssDNA binding protein.The expression efficiency of pSW4 was found to significantly higher than pSW2?10.7-fold at 4??by using GFP as reporter gene.Moreover,the Sps01203 gene in the deep-sea bacterium Shewanella psychrophila WP2 were successfully expressed using Shewanella piezotolerans WP3 as expression host and pSW4 as expression vector.Subsequent assay indicated that the protein possess nuclease activity.Specifically,Mg²⁺is essential for the ssDNA substrate,while Mg²⁺or Mn²⁺is required for the dsDNA substrate.A cold-induced expression vector pSW4 has been successfully constructed,and the applicability has been verified.Thus,our study will contribute to the further applied research of bioremediation bacterium.