Figure 8. Activity assays for At1g74130 splice variant proteins using transgenic bacteria and as exogenous additives to “Superbug” bacteria.

( A) Bacterial cells expressing one of the three different splice variant proteins were grown on LB-ampicillin plates from 1.0 mg/mL to 1.75 mg/mL (labelled L, S, and M, as in the previous figures). ( B) Graphical representation of the survival levels observed for each of the plates shown in panel A (reflected as the numbers of colonies growing). The labelling is the same as in panel A. ( C) Expression of At1g74130 variant proteins in bacteria alters production and secretion of β-lactamase. Immunoblot images of β-lactamase protein samples are shown for different bacterial strains grown in different ampicillin concentrations. The lanes contain lysates from control cells containing pET20b only (labelled Control), and one of the At1g74130 variants, L, S, and M (labelled as Variant L, S, or M). The smaller-sized bands marked “M” represents the mature β-lactamase form (29 kDa) and the larger-sized bands marked “P” represents the precursor β-lactamase form (31.5 kDa). The full immunoblot image used is provided in Figure S10. ( D) Recombinant At1g74130 variant proteins were tested for activity (enhanced sensitivity to ampicillin in this case) as exogenous additives in the same manner as that shown for yeast in Figure 7. Cells (resulting colonies) surviving the different treatments are depicted as Percent Survival in the graph. The error bars represent standard deviations (n=3). The Percent Survival was calculated relative to the No Treatment control cell numbers. “No treatment” represents the bacterial culture used (diluted to the prescribed cell number tested as described in Methods). The variant protein being tested is labelled as in the panel A. DMSO indicates the use of DMSO (5% v/v) as the delivery agent. The Mock Treatment contains all components used except with no recombinant proteins added. All treatments involve exposure to 1.25 mg/mL ampicillin.