Fig. 4.

Assessment of IFDC1 for markerless mutagenesis. IFDC1 was used to assemble a construct for the in-frame deletion of nlmA. (A) Five erythromycin-resistant clones were PCR amplified to confirm the insertion of the IFDC1 cassette. The expected PCR amplicon from the wild type is approximately 2.2 kb, while the expected amplicon from the mutant is approximately 4 kb. (B and C) Mutant strains confirmed to contain the IFDC1 cassette were subjected to a second transformation reaction to remove the IFDC1 cassette and create the markerless in-frame deletion mutation. The results of successive dilutions of that second transformation reaction mixture after it was spotted onto BHI plates (B) and BHI plates containing p-Cl-Phe (C) are shown. The UA159 parent strain was included for comparison. The dilution level is indicated in each image. This experiment was performed 3 times with similar results. WT, wild type.