Development of inducer-free expression plasmids using IPTG-inducible Pspac promoter for Bacillus subtilis

Phuong Thi Bich Chu^{1, 2, 3, 4}, Hanh Thi Thu Phan^{1, 2}, Hoang Duc Nguyen^{1, 2, 4*}, Trang Thi Phuong Phan^{1, 2, 5*}

¹Center for Bioscience and Biotechnology, University of Science, Vietnam National University, Ho Chi Minh city

²Vietnam National University, Ho Chi Minh city

³Faculty of Pharmacy, Ho Chi Minh City University of Technology (HUTECH), Ho Chi Minh city

⁴Department of Microbiology, University of Science, Vietnam National University, Ho Chi Minh city

⁵Laboratory of Molecular Biotechnology, University of Science, Vietnam National University, Ho Chi Minh city

Received 1 December 2019; accepted 3 February 2020

 

Abstract:

An inducer-free expression vector for the low expression levels in Bacillus subtilis (B. subtilis) is necessary for fundamental research. In this study, we constructed inducer-free expression plasmids carrying Pspac, a well-known IPTG-inducible promoter, by removing a part of the lacI gene. Then, we analysed the expression of the target genes bgaB and gfp⁺ in B. subtilis. Western blot experiments demonstrated that the reporters from the inducer-free plasmids with Pspac could be produced at low levels in B. subtilis strains and were equivalent to their corresponding inducible constructs with 1 mM IPTG. The reporter activities showed that inducer-free expression from the Pspac promoter was dramatically less than that of the inducer-free plasmids with the strong promoters Pgrac01 and Pgrac100 reported previously, about 16.2 to 20.3 times for BgaB and 24.7 to 34.3 times for GFP⁺, respectively. In conclusion, the inducer-free expression vectors carrying Pspac promoters allow the constitutive expression of heterologous recombinant proteins at low levels in B. subtilis.

Keywords: Bacillus subtilis, inducer-free, pHT vector, Pspac, weak promoter.

Classification number: 3.5