Benchtop and microcentrifuge preparation of Pseudomonas aeruginosa competent cells.

Although indispensable for the introduction of DNA fragments manipulated in vitro into bacteria, transformation using chemically prepared competent or electrocompetent cells is still a rather slow and labor-intensive technique, and apart from waiting for transformants to appear on selective plates, the preparation of competent cells is the slowest part of the whole process. Most published transformation protocols for use with Pseudomonas aeruginosa are modifications of protocols developed for E. coli and either involve electrocompetent cells (1–4,9,12) or cells made competent by chemical treatment (8,10). Conventional chemical transformation of P. aeruginosa has many drawbacks, most notably low transformation efficiencies and/or time-consuming procedures. Therefore, electroporation has become the method of choice for this bacterium. However, the preparation of low-ionic strength DNA and competent cells for electroporation is still a critical and relatively time-consuming process. Here we describe the adaptation of a rapid benchtopand microcentrifuge-based method previously reported (14) for obtaining low-efficiency competent E. coli cells to prepare highly competent P. aeruginosa cells for chemical transformation. For P. aeruginosa, this method yields transformation efficiencies that are comparable to those observed when using electroporation. For preparation of competent P. aeruginosa cells, strain PAO1 cells were grown in 4 mL LB broth (Difco Laboratories, Detroit, MI, USA) at 37°C until they reached saturation (usually overnight). All of the following steps, except the centrifugations, were performed with the tubes sitting on ice. A 1.5-mL microcentrifuge tube was pre-chilled on ice for 3–5 min. Aliquots (1 mL) of a stationary phase or overnight P. aeruginosa culture were transferred to the chilled microcentrifuge tubes, and the cells were harvested at room temperature by a short centrifugation for 30 s at approximately 13 000× g. Centrifugations longer than 30 s are not recommended because they cause significant reduction of transformation efficiencies. The cell pellets were resuspended with a pipet tip in 1 mL cold (approximately 4°C) 0.1 M MgCl2. The cell suspension was again centrifuged at approximately 13 000× g for 30 s at room temperature. The supernatant was removed, and the cell pellets were resuspended with a pipet tip in 1 mL cold transformation salts with glycerol (TG salts) solution (75 mM CaCl2, 6 mM MgCl2, 15% glycerol). The cell suspensions were kept on ice for 10 min and then centrifuged at approximately 13 000× g for 30 s at room temperature. After decanting the supernatant, the pellets were then resuspended with a pipet tip in 200 μL cold TG salt solution and kept on ice until use. At this stage, the cells were ready for transformation. The transformation efficiency of P. aeruginosa cells prepared using this procedure was determined using the 4.2-kb broad-host-range plasmid pUCP20T (11). Aliquots (100 μL) of the competent cells were transferred to thin-walled 13 × 100 mm borosilicate glass tubes (VWR Scientific, South Plainfield, NJ, USA) that were prechilled on ice. Next, 2–5 μL aliquots containing 100–1000 ng pUCP20T DNA that has been purified using the Qiagen® Midiprep kit (Qiagen, Valencia, CA, USA) and suspended in either 10 mM Tris-HCl (pH 8.5) or sterile water were added to the cells (DNA in this buffer or water yielded the same transformation efficiencies). A negative control received sterile water instead of plasmid DNA. This DNA-cell mixture was incubated on ice for 15 min. The mixture was then heat-shocked at 37°C for 2 min, and 500 μL LB broth were immediately added. The tubes were incubated at 37°C for 1 h in a shaking incubator. After incubation, a 200-μL aliquot of each cell suspension was plated on LB agar containing 200 μg/mL carbenicillin (Gemini Bio-Products, Woodland, CA, USA). The remaining contents of each tube were transferred to microcentrifuge tubes, and cells were harvested by centrifugation at approximately 13 000× g for 1 min at room temperature, resuspended in 200 μL LB medium, and then spread on a LB-carbenicillin plate. The plates were incubated at 37°C for 20–24 h before colonies were counted. Using the procedures described above for the preparation and transformation of competent P. aeruginosa PAO1 cells (Figure 1), we obtained 1 × 105 transformants/μg pUCP20T DNA (Table 1), using an optimal DNA concentration of 200–400 ng. This transformation efficiency is comparable to the 1.1–1.5 × 105 transformants/μg DNA when using most electroporation protocols (1–4,9,12). Only two other reports indicated higher transformation efficiencies [i.e., 1.2 × 106 transformants/μg DNA by using chemically prepared competent cells and a heat shock at 50°C (8) and approximately 2 × 106 transformants/μg DNA by employing an electroporation procedure (3)]. Although the transformation efficiency of competent cells prepared using our procedure is about 10-fold lower when compared to the most efficient procedures, it is comparable to those achieved with most published methods and therefore more than adequate for most laboratory applications. However, Benchmarks