Preparation of Bacillus subtilis cell samples and generation of an SDS-PAGE

Bacillus subtilis is a critical host for producing recombinant proteins. However, the SDS-PAGE process, including the sample preparation steps, varies among B. subtilis-related studies, making it impossible to compare findings. Hence, this paper provides a simple guide to culture and collect B. subtilis cells through an OD600 measurement and a protocol for SDS-PAGE. These techniques were applied to check the expression of a BgaB, a reporter protein and LukF-PV, a potential vaccine candidate against S. aureus, in the cytoplasm of B. subtilis under the control of a strong promoter, P-grac212. This protocol could be helpful for scientists in preparing samples and generating an SDS-PAGE experiment, as well as favoring the unification of research about protein expression in B. subtilis. METHOD SUMMARYBacillus subtilis strains were cultured in LB medium to the mid-log growth phase (OD600 reaching 0.8-1). Aliquots, equivalent to an OD600 of 2.4, were collected in a 1.5-ml tube and the cells were obtained by centrifuging at 13,000 g for 5 min. The sample preparation began by resuspending the collected cells in a lysis buffer with lysozyme. After mixing with the sample loading buffer and centrifuging, an appropriate volume of each sample supernatant was added to each well of a polyacrylamide gel. The SDS-PAGE was performed following a detailed protocol. The gels were stained, destained, photographed by a scanner and analyzed by densitometry using software.