Transformation_of_Pseudomonas_fluorescens_SBW25_by_Electroporation

Using the small (1 µl) end of a sterile Combi Loop, generously streak P. fluorescens SBW25 from a glycerol stock onto LB agar plates and incubate overnight at 30°C

• Aim to streak out cells so that nearly confluent growth is obtained (i.e. NOT single, isolated colonies).
• Prepare one streak plate for each transformation that you plan to do.
• Use antibiotic-free LB agar plates for WT cells

For each plate, use the large (10 µl) end of a sterile Combi Loop to gently scrape the cells from the plate and transfer to a sterile, 2.0 mL microcentrifuge tube containing 1 mL sterile 15% glycerol.

• Cells can be picked up efficiently by starting at the top of the plate and running the loop back and forth over the surface.

Resuspend the cells by gentle vortexing, and pellet by centrifuging at 10,000 rpm for 1 minute (increase speed/time if supernatant remains cloudy).

Remove the supernatant by pipetting, avoiding the cell pellet.

• Wash the pellet three more times with 1 mL 15% glycerol.

Resuspend the final pellet in 100 ul 15% glycerol.

Each pellet provides one transformation.

Label and chill electroporation cuvettes on ice.

Dilute DNA to be electroporated to a final volume of 5 ul. Add to cell suspension and flick gently to mix. Place DNA/cell mixture on ice.

• To avoid arcing, it is important to keep the salt concentration of DNA preps as low as possible. DNA preps should be eluted with nuclease-free water. All DNA dilutions should be made with nuclease-free water.
• For replicative plasmids use 10 - 50 ng DNA.
• Use 500 ng DNA for nonreplicative plasmids.
• IMPORTANT CONTROL: always prepare one negative control with cells + water only to check for background colonies and stability of antibiotic on the plate.

Turn on electroporator (Bio-Rad GenePulser Xcell or equivalent).

Settings (On GenePulser select Preset Programs>Bacterial Cells>P. aeruginosa)

A	B
Voltage (V)	2500
Capacitance (uF)	25
Resistance (ohms)	200
Cuvette (mm)	2

Transfer cell/DNA suspension to cuvette on ice with pipette.

Tap the cuvette a few times to release any air bubbles.

Wipe dry with Kim-Wipe and place in Electroporation chamber; close lid

Electroporate; note the time constant.

Immediately add 1 mL S.O.C to the cuvette and pipette gently to mix.

Use pipette to transfer the suspension to a sterile culture tube.

Incubate for 1-2 hours at30°Cwith shaking at 200 rpm

Prewarm LB-agar plates containing appropriate antibiotic for plasmid under selection.

For replicative plasmids plate 20 - 100 ul of the cell suspension using a sterile cell spreader.

• To ensure that single colonies are obtained, the spreader can be run over a second plate.

For non-replicative plasmids, transfer the culture to a sterile 1.5 mL eppendorf tube and collect the cell pellet by centrifugation. Remove all but 100 ul of culture medium and resuspend the cells. Plate the entire cell suspension.

• Allow residual medium to absorb into plates before flipping and placing in incubator at 30°C

Incubate plates for 1-2 days to develop the colonies

Antibiotic Concentrations for P. fluorescens SBW25 P. fluorescens SBW25

A	B
Tetracycline	20 ug/ml
Gentamycin	20 ug/ml
Kanamycin	50 ug/ml