FIGURE 6.
Design of the time-delayed in vivo assembly system and its different states during cell growth. ΔatpB-C strain DK8 was transformed with three plasmids bearing different resistance genes and different origins to gain compatibility: (i) a pBAD33 derivative (p15A ori; CmR) with structural genes of the atp operon carrying an early stop codon (δY11end) in atpH (atpBEFH*AGDC) under control of ParaBAD; (ii) a pET-22b derivative (pMB1 ori, ApR) carrying the atpH gene under control of the IPTG-inducible T7lac promoter using the weak start codon TTG for atpH; (iii) a pSC101 derivative (KanR) containing T7 gene1 under control of the IPTG-inducible T5N25lac promoter, encoding the RNA polymerase specific for promoters of phage T7. Left, cells were inoculated with OD = 0.05; ParaBAD was induced by arabinose to allow expression of the atpBEFH*AGDC genes, and the FOF1 subunits except for subunit δ (FOF1−δ) were synthesized. During this growth phase, the lac operator-controlled promoters are completely repressed. Middle, at OD = 0.3, ParaBAD is repressed by the simultaneous addition of glucose and d-fucose, and after further growth for 20 min, the atp mRNA present within the cell is completely degraded. Due to this time delay, the de novo biosynthesis of FOF1 subunits is prevented. Right, time-delayed IPTG induction of lac operator-controlled T7 and T5N25 promoters for 1 h enables the synthesis of subunit δ assembling together with the preformed FOF1 subcomplexes ab2 and c10α3β3γϵ (FOF1−δ+δ), a functional FOF1 complex. Red cross, repressed or uninduced state of promoters.