Therefore, gene disruption procedure of the fkbN gene was aided by the introduction of a kanamycin resistance cassette in order to simplify the otherwise laborious identification of secondary recombinants. In order to introduce the kanamycin resistance cassette, the Rapamycin cost pDG7 plasmid containing the fkbN flanking regions was digested using NdeI, blunt-ended and dephosphorylated. A 1323 bp blunt-end fragment containing the kanamycin resistance cassette was excised from the SuperCos 1 cosmid vector (Stratagene)

and then ligated into the vector, resulting in pDG8 (Table 1). The disruption plasmids pDG5, pDG6 and pDG8 were then introduced into electrocompetent E. coli strain ET12567 containing the conjugative plasmid pUZ8002 [32, 43]. The conjugation procedure was carried out as described previously [42]. Exconjugants were grown at 28°C on ISP4 sporulation PLX3397 mouse medium with addition of apramycin (pKC1139). Exconjugants were then inoculated into VG3 medium and cultivated at 28°C and 220 rpm to obtain a good seed culture [30]. After 24 hours, the cultures were reinoculated into a new tube with fresh

VG3 medium and cultivated at 37°C. Above 34°C the pKC1139-based vector is unable to replicate and is forced to integrate into the S. tsukubaensis genome via homologous regions, thus yielding primary recombinants. The cultures were then further subcultivated at 37°C several times in VG3 medium and then plated onto the ISP4 sporulation selleck chemicals llc medium. Harvested spores were filtered and serial dilutions were plated onto the sporulation medium without apramycin (with kanamycin in the case of fkbN disruption). 4��8C After 5–8 days of cultivation at 28°C single colonies were replica-plated onto plates without antibiotic and plates with apramycin (both with kanamycin in the case of fkbN). Primary recombinants were still resistant to apramycin, while secondary recombinants lost apramycin resistance. The apramycin sensitive colonies were

further screened using PCR to confirm the deletion. In the case of fkbN, the final screening step was simplified by the addition of kanamycin to the medium which precluded the growth of revertants to wild-type after secondary recombination, which greatly reduced the time and effort required to screen for correct secondary recombinants using PCR. After the stable secondary recombinants were identified and verified by PCR a double mutant was additionally generated in which both the fkbR and fkbN genes inactivated. Taking the ΔfkbR strain as the starting point we disrupted the fkbN gene using the same procedure as described above. Finally, all mutant strains were tested for FK506 production. Figure 2 Schematic representation of disruption plasmids and inactivated fkbN (A) and fkbR (B) genes after secondary recombination. Evaluation of the promoter activity of the selected genes from the S.