Figure 7
Reconstitution of the λcI repressor function through CpxR homodimerization. Fusion of CpxRn variants to the C-terminus of λcI1-131 (auburn brown shade) were generated. When expressed in E. coli JM109, growth was assessed by spotting 5 μl of 10-fold serially diluted (100, 10−1, 10−2, 10−3, 10−4 and 10−5) exponentially grown cultures onto LB agar containing 10 μg/ml tetracycline (Tet) and 0.1 mM IPTG (A). The assay was controlled through the expression of inactive λcI1-131 alone [vector; pKWY2428] or the dimerization-competent fusion λcI1-131-YycF120-235 [YycF(120C); pKWY-YycF(120C)][36]. Protein lysates were also fractionated on 12% acrylamide SDS-PAGE and analysed by western blotting (B). Fusions of λcI1-131 to the N-terminus of CpxRn variants were detected with rabbit polyclonal anti-CpxR antiserum. Samples were also probed with antiserum raised in rabbit and specific for chloramphenicol acetyltransferase (anti-CAT) to confirm the loading of an equal quantity of protein in each lane. The asterisks (*) highlight unknown protein bands that cross-react non-specifically with antibodies in the sera or, in some cases may represent a degradation product of the recombinant fusion proteins. Shown to the left is the approximate mobility of molecular weight standards (PageRulerTM Plus Prestained Protein Ladder, Thermo Scientific).