FIG 5.
Deletion of a DNA fragment flanked by PSLs with the IntXO recombinase produced in a pXO1-negative strain. (A) Ames 33 containing pSCP(Ω-km) was transformed with pMRmut-PI at 30°C with double selection (erythromycin and spectinomycin). After two passages at 37°C on plates containing medium with erythromycin, clones sensitive to both kanamycin and spectinomycin were selected. (B) The control primer pair PSCP1F/PSCPR was used to verify the Ω Kmr cassette deletion. The sizes of the PCR fragments are shown above the arrows, identifying the fragments before and after the deletion. Mr, GeneRuler DNA ladder mix for size determination (as in Fig. 1B). (C) Sequences of both PCR fragments obtained from pSCP(Ω-km) and pSCPΔ(Ω-km). The directions of sequencing by use of the corresponding primers are indicated by arrows. The same result was obtained with the pIntPAS, pZR1, or pZR2 plasmid. See the explanation in the text.