Underlined sequences are the sequences of the new codons used for constructing mutant Site-directed PCR mutagenesis used the internal F-R9K and R-R9K primers with the sequence mismatch CGC→AAG, causing the R9K substitution. The same procedure was applied to generate the second mutation using the internal mismatched primers F-E129G and R-E129G, to generate the sequence GAA→GGG, causing the E129G substitution. The resulting fragment was digested with XbaI and KpnI and inserted into pSK53 cut with the same enzymes to obtain plasmid pSK5S13-9

K-129 G (Figure 1B). This was digested with SacI and BglII and the recovered fragment was ligated

into pSS4245 cut with SacI and BamHI. After transformation into E. coli SM10, the resulting plasmid was designated as pSS5S13-9 K-129 buy KU55933 G. Allelic exchange to insert the modified S1 gene back into its original location in the B. pertussis chromosome was performed as above but without selection of the exconjugants by chloramphenicol. The desired strains in this case have lost this marker and therefore screening by replica plating Verubecestat was necessary to identify colonies with the desired phenotype CmS and SmS. The resulting Tohama derivative was designated as Bp-WWC (Figure 2B). The integration of the S1 mutated gene at the designated position was confirmed by PCR with the specific primers. The primers could bind the upstream 5′ (5′F-int and R-R9K primers), 3′ (F-E129G and 3′R-int primers) downstream flanking regions, and inside the S1 gene. {Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|buy Anti-infection Compound Library|Anti-infection Compound Library ic50|Anti-infection Compound Library price|Anti-infection Compound Library cost|Anti-infection Compound Library solubility dmso|Anti-infection Compound Library purchase|Anti-infection Compound Library manufacturer|Anti-infection Compound Library research buy|Anti-infection Compound Library order|Anti-infection Compound Library mouse|Anti-infection Compound Library chemical structure|Anti-infection Compound Library mw|Anti-infection Compound Library molecular weight|Anti-infection Compound Library datasheet|Anti-infection Compound Library supplier|Anti-infection Compound Library in vitro|Anti-infection Compound Library cell line|Anti-infection Compound Library concentration|Anti-infection Compound Library nmr|Anti-infection Compound Library in vivo|Anti-infection Compound Library clinical trial|Anti-infection Compound Library cell assay|Anti-infection Compound Library screening|Anti-infection Compound Library high throughput|buy Antiinfection Compound Library|Antiinfection Compound Library ic50|Antiinfection Compound Library price|Antiinfection Compound Library cost|Antiinfection Compound Library solubility dmso|Antiinfection Compound Library purchase|Antiinfection Compound Library manufacturer|Antiinfection Compound Library research buy|Antiinfection Compound Library order|Antiinfection Compound Library chemical structure|Antiinfection Compound Library datasheet|Antiinfection Compound Library supplier|Antiinfection Compound Library in vitro|Antiinfection Compound Library cell line|Antiinfection Compound Library concentration|Antiinfection Compound Library clinical trial|Antiinfection Compound Library cell assay|Antiinfection Compound Library screening|Antiinfection Compound Library high throughput|Anti-infection Compound high throughput screening| Insertion of a second set of the 5 PT structural genes The sequences flanking the targeted insertion site (Figure 3A) were first cloned to obtain pSKPD5Cm3. The upstream 1688 bp fragment was amplified with the primers 5′F-PD-ApaI and 5′R-PD-MCS, digested with ApaI and KpnI,

and ligated into pSK5Cm3 cut with the same enzymes to yield pSKPD5′-Cm. The downstream 2980 bp fragment was amplified with the primers 3′F-PD-MCS and 3′R-PD-BglII, digested with ifoxetine XbaI and BglII, and ligated into pSKPD5′-Cm cut with the same enzymes. The resulting plasmid was designated as pSKPD5Cm3 (Figure 3B). The conjugative construct was obtained by digesting this plasmid with NotI and BglII and ligation into pSS4245 which was digested with NotI and BamHI, resulting in plasmid pSSPD53-Cm. Conjugative transfer and selection for SmS and CmR provided the desired B. pertussis derivative Bp-PD53-Cm, where the presence of the intact upstream, downstream, and CmR insert was confirmed by PCR amplification.