Cellulose ester filters

The sgRNA-pDNA library was introduced into the FQR pump hyper-expressors and WT strain with chromosomally encoded Tet-inducible dCas9 via electroporation. About 50ul electrocompetent cells were transformed with 23–46ng of the pDNA, the resuspended mixture diluted 1/10, and 200µl plated on five large pre-warmed SOC agar plates overlain with mixed cellulose ester filters (0.45µm pore size, 137mm diameter, Advantec). The mixture was spread on the filters using beads. This was repeated for a total of 3 transformations per strain background. These transformants were allowed to recover at 37°C for an hour on SOC agar plates before the filters were transferred to selective LB agar plates containing 50µg/mL Carb and incubated overnight at 37°C for about 13.5–14 hours. The following day, filters were pooled to acquire somewhere around 250,000–350,000 CFUs, estimated based on using image analysis of previous pools (ImageJ). Pooling entailed washing colonies off with 100mL sterile PBS with gentle shaking in a sterile round bottom 3L beaker. Glycerol was added to a final concentration of 10–20% and 1mL aliquots were placed into cryotubes for −80°C storage until needed.

Transposon banks with Himar-1 transposon were created as described (17 , 36 (link)). 50μl electrocompetent A. baumannii cells were electroporated with 300ng pDL1100 (Himar-1-Kan^R). Cells were recovered in 1mL SOC broth, placed in 14mL snap cap tubes (Corning) and aerated on a drum roller for 15min at 37°C. The cells were pelleted and resuspended in 400μl SOC, then spread onto two 150 mm culture dishes containing mixed cellulose ester filters (0.45μm pore size, 137mm diameter; Advantec) that had been placed on top of the SOC agar. Plates were incubated for 1hr at 37°C before filters were transferred to 150 mm culture dishes containing LB+20μg/mL kanamycin and incubated overnight (~16hrs) at 37°C.
Mini-Tn10 banks were also made in the GPN strain background. GPN electrocompetent cells were transformed with 75ng pDL1073 (mTn10-neo), with subsequent steps following the same procedure as used for Himar-1. CFU counts were determined from plate images using an Image J counting routine. Mutants were pooled from filters by gentle shaking in 50–75mL sterile PBS. Glycerol was added for a final concentration of around 10% and aliquots stored at −80°C until use. About 21 independent subpools were created in each strain background (producing around 100,000–150,000 cumulative unique mutants).