Construction of cold inducible expression vector pSW4 and expression of DNA polymerase III epsilon subunit

Abstract:

[Objective] Deep-sea derived shutter vector pSW2 has been constructed previously. In this study, a cold inducible expression vector was constructed based on pSW2 to provide useful explant materials and toolbox for the construction of an engineered strain that can degrade environmental pollutants. [Methods] Mutation experiments were performed to identify essential gene in the vector, and green fluorescent protein (GFP) was used as reporter gene to test the expression efficiency of mutated vectors. Finally, the cold inducible vector pSW4 was constructed by adding purification tags and multiple cloning sites, and by replacing of the promoter region. [Results] 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 higher than that of pSW2 (10.7-fold at 4 °C) by using GFP as reporter gene. The sps01203 gene in the deep-sea bacterium Shewanella psychrophila WP2 was successfully expressed using Shewanella piezotolerans WP3 as expression host and pSW4 as expression vector. Subsequent assay indicated that the protein possesses nuclease activity. Specifically, Mg2+ is essential for the ssDNA substrate, whereas Mg2+ or Mn2+ is required for the dsDNA substrate. [Conclusion] A cold-induced expression vector pSW4 has been successfully constructed, and its applicability has been verified. Thus, our study will contribute to the further applied research of bioremediation bacterium.

Key words:Cold induction, Vector construction, Deep-sea bacterium, Bioremediation