The Periplasmic Protein TolB as a Potential Drug Target in Pseudomonas aeruginosa (original) (raw)
Figure 1
Scheme of the strategy used to generate the P. aeruginosa PAO1 tolB conditional mutant.
An exogenous copy of the tolB coding sequence under the control of an arabinose-dependent promoter was inserted into the attB neutral site of the P. aeruginosa chromosome, by using the integration-proficient plasmid mini-CTX1-araC_PBAD_tolB (Table 1). After Flp-mediated removal of the mini-CTX1 backbone (not shown), the resulting strain (PAO1 araC_PBAD_tolB) is a merodiploid for tolB. In-frame deletion of the endogenous copy of tolB was obtained using the suicide plasmid pDM4Δ_tolB_ (Table 1). Sucrose selection was carried out in the presence of arabinose to select removal of the pDM4 backbone, followed by PCR screening to identify clones carrying the tolB in-frame deletion. One of these clones was selected and used for the following analyses. This conditional mutant was named PAO1 Δ_tolB araC_PBAD_tolB_.