Construction of pON.mCherry.

<p><b>(A)</b> Promoter regions of pXDC50 (parental plasmid) and pON.mCherry. DNA fragments from a mutagenic PCR strategy targeting the lac repressor-binding site were cloned into pXDC50 as described in the Materials and Methods. Black circles identify nucleotide changes between the parental pXDC50 <i>lacO1</i> operator and the mutated operator in pON.mCherry. <b>(B)</b> pON.mCherry plasmid map. Unique restriction sites are shown; R, EcoRI; N, NdeI; X, XbaI; Sa, SalI; Sp, SphI; H, HindIII. <b>(C), (D)</b> Screening pON.mCherry library in <i>L</i>. <i>pneumophila</i> demonstrates a variety of promoter strengths in the pON mutant operator library.