Figure 3

Comparative growth and fluorescence of colonies with the pGLO, pLL2522 and pLL 2524-XXX plasmids. (A) Contrasting growth patterns of colonies of XL1 Blue cells of E. coli (Stratagene Inc.) transformed with (a) the pGLO plasmid and (b) the pLL2522 (IS2orfAB::GFP) plasmid. Cells were plated on lysogeny broth (LB) plus carbenicillin and arabinose, incubated at 37°C for 48 hours and irradiated with UV light. (B) XL1 Blue cells transformed with the ligation products generated by cloning PCR products recovered from the Genemorph II Random mutagenesis of IS2orfAB DNA, into the Eco RI/Nhe I sites of pGLO-ATG2. Colonies were generated as described above and viewed after 72 hours at 37°C. Arrows identify the faster growing more brightly fluorescing colonies, the vast majority of which contained plasmids pLL2524-XXX (IS2orfAB::GFP-GMF) with loss-of-function mutations in the orfAB gene. Isolated colonies at the periphery of the Petri dish (see white asterisk) occasionally produced false positives without mutations or with silent mutations, for example, A42T. PCR: polymerase chain reaction.