In addition to the tellurite-resistance marker, pMo130-TelR https://www.selleckchem.com/products/mk-4827.html also carries a kanamycin-resistance marker, the reporter gene xylE which converts pyrocathechol to a yellow-colored 2-hydroxymuconic semialdehyde,
and a modified sacB gene . Next, DNA fragments of approximately 1 kb upstream and 1 kb downstream of the target region to be deleted was ligated with linearized pMo130-TelR give pMo130-TelR-(Up/Down) (Figure 1A). Figure 1 Strategy for deleting adeL-INCB028050 adeFGH and adeIJK operons in MDR A. baumannii DB and R2. Panel A, The upstream (UP) and downstream (DOWN) regions (approximately 1 kb) flanking the target genes was cloned into the suicide vector, pMo130-TelR. pMo130-TelR was constructed by inserting a 3.26 kb XmaI-digested tellurite-resistance cassette from pwFRT-TelR into the XmaI site of pMo130. Recombinants obtained after first cross-over were selected for inheritance of tellurite-resistance and xylE + (yellow colonies). These recombinants also do not produce any amplimers with the primer pair pMo130Tel F and pMo130Tel R. During the second cross-over, mutants with gene deletion (1) were selected
SN-38 manufacturer for loss of sacB by passaging the first cross-over recombinants in media containing sucrose. The second cross-over could also yield parental genotype (2). Deletion of the adeFGH operon (Panel B) and the adeIJK operon (Panel C) showing the positions of the respective UP and DOWN fragments flanking each deletion (striped and hatched boxes, respectively). The locations of the PCR primers used for amplifying
the UP and DOWN fragments and for qRT-PCR analysis of gene expression are indicated by black arrows while P1, P2 and Nutlin-3 manufacturer P3 (grey arrows) are the locations of predicted promoters for adeFGH operon, adeL, and adeIJK operon, respectively. To construct the suicide plasmid for deletion of adeFGH, a 1 kb DNA fragment located upstream of adeF was amplified from R2 genomic DNA using the primer pair: AdeGUp(Not1)F and AdeGUp(BamHI)R (Figure 1B). The amplimer was digested using Not1 and BamHI and inserted into pMo130-TelR, creating pMo130-TelR-adeFGH(UP). Next, another 1 kb fragment located downstream of adeG was amplified using the primer pair: AdeGDwn(BamHI)F and AdeGDwn(Sph1)R and cut with BamHI and SphI, and inserted into pMo130-TelR-adeFGH(Up), thus creating pMo130-TelR-adeFGH(Up/Down) (Figure 1B). The plasmid construct was first introduced in E. coli S17-1 and subsequently delivered into A. baumannii R2 and DB by biparental conjugation. A. baumannii transconjugants (first crossovers) were selected on LB agar containing 30 mg/L tellurite and 25 mg/L gentamicin. These tellurite-resistant colonies which carry genomic insertion of pMo130-TelR-adeFGH (Up/Down) produced yellow colonies when sprayed with 0.