FIG. 1.

Assembly of an IPTG-inducible promoter. (A) Schematic drawing showing the pilA promoter from −855 to 1 bp inserted in front of a leader sequence containing the lac operator for binding of and repression by the lac repressor LacI. The KpnI and EcoRI restriction sites were used to insert a gene of interest (often pilA). (B) Base sequence of the leader and the lac operator. The sequence is preceded by the −24 and −12 boxes of the σ⁵⁴-dependent pilA promoter and the transcriptional start site at position 1. The locations of the lac operator and the RBS are indicated. The translational start site ATG is indicated by boldface type. (C) Insertion of the inducible promoter into the M. xanthus chromosome by homologous recombination within the pilA locus of DK10410. After electroporation of the plasmid into M. xanthus cells, a single homologous crossover produced a tandem duplication of the pilA promoter in which the upstream chromosomal region (thick line) drove the expression of the inducible promoter, whereas the 855-bp upstream region cloned in the vector (thin line) drove the expression of the native pilA gene, most often a ΔpilA allele.