Arabinose was added to a final concentration of 10 mM. In mating experiments, exconjugant P. aeruginosa PAO1 clones were selected on PIA (Difco) containing Cb. Construction and screening of PAO1 shotgun antisense libraries Genomic DNA was isolated from P. aeruginosa PAO1 using an illustra GenomicPrep Cells
and Tissue DNA Isolation Kit (GE Healthcare). DNA was diluted in 10 mM TE buffer (pH 8.0) and nebulized to obtain sheared fragments spanning 200–800 bp (Additional file 1: Figure S1A). Following ethanol precipitation, fragmented DNA was treated with nuclease BAL-31 and Klenow (New England Biolabs) for 10 min at 30°C to obtain blunt ends. After enzyme inactivation with 1 mM EDTA, DNA was dialyzed against 20 mM Tris–HCl (pH 8.0). pVI533EH and pHERD20T were digested with SmaI (New England Biolabs) and dephosphorylated using shrimp alkaline AZD1152 purchase phosphatase (Roche). Fragmented DNA was ligated to dephosphorylated vectors using T4 Ligase
selleck screening library (Takara Bio) at 16°C overnight. Ligation mixtures were transformed into E. coli JM109 by electroporation, and transformants were selected on LB plates supplemented with Cb. The resulting transformant colonies composing the SAL were arrayed and cultured in 96-well microplates. Quality control by PCR of single colonies, using primers flanking the multi-cloning site (Additional file 1: Figure S1B), was performed to check the presence and the size of a genomic insert. SALs were mobilized from E. coli to P. aeruginosa PAO1 by conjugative triparental mating. E. coli donor strains were grown overnight in 96-well next microplates in LB broth supplemented with Cb. The recipient P. aeruginosa PAO1 and helper E. coli HB101/pRK2013 strains were grown overnight in flasks in LB broth. Thirty microliters each of helper, recipient, and donor strains were mixed in microplate wells. After mixing, microplates were centrifuged at 750 × g for 5 min and Selonsertib incubated for 3 h at 37°C. Cell pellets resulting from triparental mating were resuspended in 90 μl of LB, and 2 μl of each mating mixture were spotted on PIA plates supplemented with
Cb, both in the absence and presence of 10 mM arabinose, to counter select E. coli donor and helper strains. Exconjugant cell spots were inspected for growth defects following 24–48 h of incubation at 37°C. The PAO1 growth-impairing inserts in pVI533EH/pHERD20T derivatives were sequenced following PCR amplification using oligo pVI533-F/pVI533-R and pHERD-F/pHERD-R, respectively (Additional file 6: Table S1). The resulting sequences were matched to the PAO1 genome at the Pseudomonas Genome Database . Acknowledgments The authors are grateful to Andrea Milani and all members of the laboratory for their helpful discussions and technical support. This work was funded by the Italian Cystic Fibrosis Research Foundation (grant FFC#10/2004) and by the European Commission (grant NABATIVI, EU-FP7-HEALTH-2007-B contract number 223670).