Deletion of the otc cluster from the genome of the ATCC 10970 strain was carried out using an iterative marker excision system (Additional file 1: Figure S15) [43]. For construction of the plasmid for deletion of the otc gene cluster, the BAC vector R3A4 from a BAC library (unpublished) was initially identified, which contained a 37 kb fragment with the entire otc gene cluster and the up and down region of the otc cluster. The antibiotic cassette (e.g., hygromycin, erythromycin) was flanked with the P-GG and B-CC sites for ΦC31 integrase, and amplified by PCR with primers HSU-OTC34 and HSU-OTC35 (Additional file 1: Table S1). Using the PCR-based λ-red recombination technique [44], the amplified hyg-ery cassette (Additional file1: Table S3) was used to delete the otc cluster on BAC R3A4 containing the entire otc gene cluster from the otrB gene to the otrA gene, on the other side of the otc gene cluster. The resulting BAC with the entire cluster substituted with the hyg-ery resistance cassette was introduced into ATCC 10970 by conjugation [39], and was selected for erythromycin resistance. Double crossover mutants were selected using blue-white [45]. Finally, the antibiotic cassette was excised from the chromosome of the selected double-crossover mutants by expression of the ΦC31 integrase from the pUWLint31 plasmid [43]. Cluster deletion was confirmed by PCR, using the HSU-OTC41 and HSU-OTC46 primers (Additional file 1: Table S1), followed by sequencing of the PCR-amplified fragment.

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