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. 2012 Oct 31;7(10):e48608. doi: 10.1371/journal.pone.0048608

Figure 1. Purification and detection of C. difficile Spo0A. A.

Figure 1

Heterologous overproduction of Spo0A-6xHis and Spo0A-DBD-6xHis in E. coli Rosetta(DE3) pLysS. M  =  molecular weight marker, numbers indicate hours after induction with 1 mM IPTG. P  =  metal affinity purified protein. Lysates were separated on a 12% SDS-PAGE. B and C. Immunoblot detection of Spo0A in total cell lysates of a C. difficile spo0A mutant (CT::spo0A) and a wild type strain (630Δerm). Times indicated range from early exponential (3 h post inoculation) to late stationary growth phase (48 h post inoculation). Sample volumes were corrected for OD600 to ensure loading of similar amounts of total cell lysate in each lane. For details see Materials and Methods. M  =  molecular weight marker. B. ECL+ detection. C. Fluorescent detection. Y-axes show peak volumes normalized to values at 48 hours post inoculation (closed diamonds; left axis) and optical density readings at 600 nM (open squares; right axis). Inset shows the blot on which the curve is based. Vertical dashed line indicates the moment Spo0A levels increase sharply (6 hours post inoculation).