Gene pulser xcell microbial system

ΔrfbC E. coli strains (Keio collection; Horizon Discovery #OEC4987-200827372) were grown overnight on LB plates with 50 μg/ml kanamycin. Colonies were inoculated from LB plates into 10 ml fresh LB broth and incubated overnight at 37 °C with shaking at 225 rpm. One milliliter of overnight culture was inoculated into 100 ml fresh LB medium with kanamycin and incubated at 37 °C with shaking at 225 rpm until A600 was approximately 0.6. Cells were harvested by centrifugation (4200g, 10 min, 4 °C). The cells were resuspended in 40 ml prechilled autoclaved Milli-Q water. Cells were harvested by centrifugation as above and resuspended in 40 ml prechilled autoclaved 10% (v/v) glycerol. A minimum of three 10% glycerol washes were conducted before cells were resuspended in 500 μl 10% (v/v) glycerol. Complementation plasmids (40 ng) were added to 50 μl electrocompetent cells, vortexed for 30 s, and incubated on ice for 10 min. The suspension was transferred to a prechilled 0.1 cm electroporation cuvette (Thermo Scientific #5510-11). A single pulse of 1.8 kV was given by a Gene Pulser Xcell Microbial System (Bio-Rad #1652662). Cells were recovered by adding 1000 μl prewarmed LB medium. The complemented cell suspension was incubated at 37 °C for 1 h shaking at 225 rpm. Transformed cells were grown on LB agar plates supplemented with 100 μg/ml ampicillin overnight at 37 °C.

Custom PNA oligomers (Table 1) were synthesized by Bio Synthesis (Lewisville, TX). Oligomers were designed complementary to the predicted Shine Dalgarno region or inclusive of the start codon. A BLASTn analysis was performed for each PNA sequence against the full genomes of each respective bacterium to confirm specificity for the sequence of interest. PNA oligomers were biotinylated using EZ-Link Sulfo-NHS-Biotin, (Thermo Fisher Scientific Inc., Waltham, MA). Labeled PNA was purified using Pierce C18 Spin Columns (Thermo Fisher Scientific Inc.) Biotin-labeled PNA was used to confirm rOmpB PNA binds to the targeted region. Dot blots were used to confirm the successful biotinylation of oligomers. To deliver, 2 μM PNA was mixed with 1 x 10⁷ purified rickettsia in a total volume of 100 μl of 250 mM sucrose in a 0.2 cm gap sterile cuvette on ice. Rickettsiae were then electroporated using a Gene Pulser Xcell Microbial System (Bio-Rad, Hercules, CA) by applying a 5 ms pulse (2500 V, 25 μF, 200 Ω). The PNA-rickettsiae suspension were transferred to a microcentrifuge tube and 400 μl of complete DMEM (5% FBS) medium was added to the electroporated rickettsia for recovery at room temperature for one hour.

Clinical E. coli was tagged with green fluorescent protein (GFP) following a previously published protocol with free use GFP b (fuGFPb) [51 ]. E. coli cells were made electrocompetent by centrifuging overnight cultures of E. coli AM-1 (5000× g, 4 °C for 15 min). The supernatant was discarded and the pellet was resuspended in 50 mL of cold sterile 10% v/v glycerol and centrifuged (4500 rpm, 4 °C for 15 min). The pellet was then resuspended in 5 mL of 10% (v/v) glycerol again to make bacteria electrocompetent and stored at −80 °C prior to transformation. Plasmid DNA containing fuGFPb and kanamycin resistance was added to 50 μL of bacteria and the mixture was transferred to an electroporation cuvette. The mixture was subjected to 2500 V, 25 µF, and 200 Ω on the electroporator (Gene Pulser Xcell Microbial System, BIO-RAD, Hercules, CA, USA), and then incubated for 1 h at 37 °C under dynamic conditions. Following this, the transformed bacteria were plated onto Luria–Bertani (LB) agar containing kanamycin to select for successfully transformed bacterial colonies.