Dandekar & Chandler:Electroporation with pseudomonas or burkholderia

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General Info About Electroporating Pseudomonas or Burkholderia

• Electroporation with non-E. coli strains, P. aeruginosa and B. cenocepacia in particular, can be hit or miss
• Efficiency of electrocompetently-prepared cells reduces drastically if frozen and used another day -- for best results make cells fresh each time
• Most referenced in the lab, with a nice and detailed protocol: mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa

Electroporating pseudomonas the quick and dirty way

from Brook

• Note: This only works well for replicating plasmids. If you are trying to get in a suicide plasmid, then you are better off using conjugation - Brook
• Makes one aliquot

1. Grow up an overnight culture of your strain in LB.
2. Spin down 1 ml of culture (3 min at 8,000 rpm)
3. Resupend cells in 1 ml of sterile 10% sucrose, spin again
4. Remove sucrose, repeat sucrose wash
5. Resupend cell pellet in 100 ul of 10% sucrose, add DNA (1 ul of a high copy plasmid mini prep works fine), electroporate on setting Ec2 for 2 mm gap cuvettes, or Ec1 for 1 mm cuvettes.
6. Add 1 ml LB, recover 1 hr at 37 degrees, plate on selective medium

Electroporating burkholderia the quick and dirty way

from Thao

• Note: This only works consistently for B. thailandensis
• Makes 1-2 aliquots
• Inoculate your cells into LB early in your day in case doubling is slow
• Turn on the centrifuge early to make sure it is at 4°C when you are ready to spin

1. Back dilute 1 mL overnight culture into 100 mL LB; grow to OD600 ~0.5
2. Chill cell flask and empty 50 mL conical tubes on ice for 20 minutes; divide cells into tubes
3. In centrifuge pre-chilled to 4°C, pellet cells for 20 min (this is way excessive -- I do 5 min - Nicole) at 5,000 rpm
4. Pour off supernatant, wash with 100 mL cold sterile water and pellet
5. Pour off super and wash again with 100 mL sterile water; pellet
6. Pour off super and wash with 2 mL cold 10% glycerol; pellet
7. Pour off super and re-suspend pellet in 200 μL 10% glycerol
8. Use 100 μL aliquots for electroporation; add plasmid to cells before transferring to pre-chilled electroporation cuvettes
9. Electroporate on setting Ec2 for 2 mm gap cuvettes, or Ec1 for 1 mm cuvettes
10. Add 1 ml LB, recover 1 hr at 37 degrees, plate on selective medium