(2003) with slight modifications. Cosmids of S. coelicolor containing the genes for replacement were introduced by transformation into E. coli BW25113 (pIJ790). Electrocompetent cells were prepared and electroporated with a PCR product (containing an aac(3)IV gene) using a GenePulser II (Bio-Rad Inc.). The PCR-targeted constructs were introduced by electroporation into E. coli ET12567 (pUZ8002) and then transferred by conjugation into S. coelicolor. Thiostrepton-resistant colonies were selected for single

crossing PD0332991 chemical structure over between the cosmid and the host chromosome. After sporulating on MS medium without antibiotic selection, thiostrepton-sensitive but apramycin-resistant colonies were screened to obtain double-crossover clones. To remove the aac(3)IV marker for the next round of gene disruption and replacement, cosmid with the aac(3)IV gene inserted in a FRT-aac(3)IV-FRT cassette was introduced by electroporation into E. coli BT340 containing a flp gene encoding Flp recombinase to remove the cassette. Clones containing a double-crossover allelic exchange in S. coelicolor were confirmed by PCR analysis and some clones (i.e. ZM4) by microarray hybridization analysis performed in the Shanghai Biochip Inc. To delete a large segment (e.g. > 40 kb)

on the S. coelicolor chromosome, two fragments (e.g. > 5 kb) from different cosmids of the ordered library plus a kanamycin resistance gene (kan) were cleaved and cloned in the polylinker of pHAQ31 or pHY642. The

resulting plasmid was introduced by electroporation into E. coli ET12567 (pUZ8002) and ADP ribosylation factor then Ixazomib cost transferred by conjugation into S. coelicolor. Thiostrepton-resistant colonies were selected for single crossing over, and thiostrepton-sensitive but kanamycin-resistant colonies for double crossing over. Clones containing a double-crossover allelic exchange in S. coelicolor were further confirmed by PCR analysis. A 2.6-kb fragment (digested with XbaI and NheI) containing a phiC31 integrase gene was cloned in a pHAQ31-derived cosmid containing the entire actinorhodin biosynthetic gene cluster. The resulting plasmid, pCWH74, was introduced by conjugation into Streptomyces strains. To quantitate the production of actinorhodin, strains were inoculated into R2YE (lacking CaCl2, KH2PO4, and L-proline) liquid medium, 1 mL culture was harvested, and spun at 15 294 g for 1 min to collect the supernatant, which was further treated with KOH and scanned at 640 nm. Measurements of actinorhodin production were carried out by the method of Kieser et al. (2000). PCR-targeting of cosmids is a precise and efficient method for gene disruption and replacement in Streptomyces. Because two long segments (e.g. > 5 kb) on a suicide plasmid are employed for homologous recombination with chromosomal sequences, high frequencies of single- and double-crossover events can usually be obtained by screening a few clones (Gust et al., 2003).