Construction of expression plasmid for Bacillus subtilis using Pspac promoter and BgaB as a reporter

Abstract

Introduction: In basic research, it is essential to use an inducible promoter which can be controlled to express a small amount of protein for studying their roles in the cell. Pspac, a well-known weak promoter for Bacillus subtilis, uses isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducer. However, plasmids carrying this promoter such as pHCMC05 still have a disadvantage which harbors a repetitive DNA fragment of about 200 bp, resulting in structural instability in Escherichia coli, causing difficulty during cloning. Methods: In this study, we constructed a plasmid that does not carry the repetitive sequences and investigated plasmid structural stability in E. coli, then measured the β-galactosidase reporter gene (bgaB) expression in B. subtilis. Results: The constructed plasmid pHT2002 was stable over 56 generations while pHCMC05-bgaB was structurally instable and ultimately lost after 42 generations. BgaB activities and Western-blot indicated that BgaB-coding gene under control of IPTG-inducible promoter Pspac could be expressed at low levels. Conclusion: The study demonstrated that the new expression plasmid without the repeated sequences retained its structural stability in E. coli facilitating the cloning step. The expression plasmid with Pspac promoter for B. subtilis could be used to express a modest amount of the heterologous protein in the presence of IPTG.