The resulting plasmid was transformed into E. coli and designated as pSKPD253. The chloramphenicol resistance gene was obtained by PCR amplification from plasmid pACYC184 using primers carrying a BamHI(CmF-BamHI) and XbaI (CmR-XbaI) restriction site. The PCR product was

digested with the two enzymes and cloned into pSKPD253 cut with the same enzymes. After ligation, the resulting plasmid was transformed into E. coli, verified by restriction analysis and designated as pSKPD25Cm3. The plasmid was digested with NotI and SpeI and the #Alisertib datasheet randurls[1|1|,|CHEM1|]# resulting fragment was ligated into pSS4245 which was doubly digested with the same enzymes. The resulting plasmid was designated as pSSP2D5Cm3 and transformed into E. coli SM10. Conjugation was conducted as described above by using Bp-WWD as the recipient B. pertussis strain with selection of CmR and SmS single colonies. The integration of Cm R gene at its designated position was confirmed by PCR with the primers that specifically bind to only the upstream 5′ (5′FPD2-int and 5′RCM-int primers), 3′ (3′FCM-int and 3′RPD2-int primers) downstream flanking regions, and inside the Cm R gene. Integration of prn gene under control selleck compound of fha promoter The structural gene of PRN was amplified from B. pertussis DNA using a primer starting at the ATG

start codon (F) and a primer carrying an XbaI (R) restriction site. The 2,808 bp amplified product containing only the coding region and the terminator was treated

by an ‘A’ tailing protocol (Promega, USA). The resulting fragment was cloned into pGEM-T easy vector to obtain a plasmid designated as pGEM-TPRN which was verified by restriction analysis. In an initial workup to create a second copy of the PRN gene driven by the stronger FHA promoter, the FHA promoter was isolated from B. pertussis DNA by PCR amplification and inserted ahead of the PRN gene. The FHA promoter was amplified by primers carrying the BamHI (FHAproF-BamHI) and a Urease polylinker containing NdeI-XbaI (FHAR-MCS). The purified product was cut with BamHI and XbaI then the recovered DNA fragment was ligated into pSKPD253 cut with the same enzymes. The resulting plasmid designated as pSKPD253Fp was verified by restriction analysis. This plasmid was cut with NdeI and XbaI, then ligated with the PCR product of the prn gene which was amplified from pGEMTPRN by PRNF-NdeI and PRNR-XbaI primers and cut with the same enzymes. The resulting plasmid was designated as pSKPD25FpPRN3 (Figure 5B). The conjugative construct was obtained by digesting this plasmid with NotI and SpeI and ligation into pSS4245 digested with the same enzymes. The resulting plasmid was designated as pSSPD2FpPRN. This construct was inserted at the selected location of the Bp-WWD chromosome to replace the chloramphenicol resistance marker introduced using the usual allelic-exchange procedures and screening as described above.