Broad-host-range application of the srfA promoter from Bacillus subtilis in Escherichia coli

Publication date: Available online 30 November 2019Source: Journal of Microbiological MethodsAuthor(s): Chengran Guan, Yan Ma, Xuan Chen, Ruifeng Zhao, Xinyuan Huang, Jianbo Su, Dawei Chen, Zhen Lu, Qiming Li, Ruixia GuAbstractThe promoter of the srf operon (PsrfA) had been used to construct a cell-density-dependent expression system in B. subtilis in our previous work. The PsrfA and its derivative PsrfA12 showed good performance of heterologous protein expression in B. subtilis. In this work, using green fluorescent protein (GFP) and β-galactosidase (LacZ) as the reporter proteins, the host feasibility and expression characteristics of the PsrfA and PsrfA12 in E. coli were identified. The prominent green fluorescence shooted by laser scanning confocal microscope, fluorescence intensity measured by spectrophotometer and the distinct protein bands detected by SDS-PAGE demonstrated that the GFP could be largely expressed under the control of the PsrfA and PsrfA12 in the E. coli host strain of BL21 (DE3) and JM109 and the expression of GFP in strain BL21 (DE3) was much higher than that of in strain JM109. Meanwhile, the promoter PsrfA 12 was much stronger than PsrfA to the extent that the GFP controlled by PsrfA12 in strain BL21 (DE3) was leaked into the supernatant. And the fluorescence intensity detected in the supernatant of the recombinant strain BL21 (DE3) containing PsrfA12 was 10.25-fold higher than that of strain JM109 containing PsrfA. Moreover, the LacZ could also be ...
Source: Journal of Microbiological Methods - Category: Microbiology Source Type: research