Figure 2.

Overview of the papillation and mating-out assays used for detection and measurement of transposition frequency of mutant Himar1 transposases. (A) Papillation assay (after ref. 43). The papillation screen was used to examine a pool of mutant transposase sequences to recover hyperactive transposases. The Himar1 coding sequence was mutagenized via error-prone PCR and was cloned into pBAD24. Transposase from this construct can mobilize a nonautonomous Himar1 transposon carrying an in-frame fusion of the 3′ ITR and a defective lacZ gene from the F plasmid to the E. coli chromosome. If the transposon fuses in the correct orientation and frame to an E. coli gene, a fusion protein can be produced with β-galactosidase activity. The subpopulation of E. coli in the colony in which this occurs are thus transformed from Lac(−) to Lac(+). When plated on MacConkey lactose agar, transposition can be scored by the formation of papillae, or red bumps, that form on the colonies. The greater the number of papillae after a given period of time, the higher the frequency of transposition. (B) Mating-out assay. The mating-out assay was used to quantify the relative frequency of individual transposase constructs. A plasmid expressing transposase isolated from the papillation screen was transformed into a strain of E. coli that contains a GenR F plasmid and a plasmid (p15 ori) carrying a KanR Himar1 transposon. Cells were plated on media to select for each plasmid. Individual colonies were grown overnight under the same selection in rich broth. The next day, these cells were mated to a NalR strain of E. coli. The total number of exconjugates were detected by selecting recipient cells with Nal-Gen. Transpositions into the target F plasmid were detected by plating the mated cells on Nal-Kan. The ratio of NalR-KanR exconjugates to NalR-GenR exconjugates is a measure of the frequency of transposition.