I am having trouble with electroporating the pCP20 plasmid into Pectobacterium wasabiae (gram- plant pathogen). I'm following the Datsenko & Wanner (2000) protocol and I need to get the antibiotic resistance gene out from the genome in order to do a double-knockout. I tried chemical transformation with E. coli DH5α strain and got 100+ and ~40-50 colonies (had 2 batches of pCP20). I know that transformation frequency in P. wasabiae is ~2-fold lower compared to E. Coli DH5α and would like some tips. I've searched for protocols for making competent cells, but so far haven't found one that works well. ATM I use the following technique: 1) grow cells over-night in 5ml LB with no antibiotic at 30 C (best for P. wasabie, cells die at higher temp) 2) add 1 ml of ON culture to 50 ml SOB medium 3) grow until OD (580 nm) reaches ~400 (took abt 3 hours) 4) put flasks on ice, wait 5-10 mins 5) centrifuge at 4C and 4000 rpm for 10 minutes 6) wash cells with 25 ml 10% glycerol 7) transfer cells to 1,5 ml eppendorf tube and repeat wash step 2 times (4000 rpm at 4C) (maybe washing is not as effective in such a small volume?) 8) remove supernatant after last wash and add ~200 ul 10% glycerol 9) transfer 40 ul to different eppendorf tubes 10) freeze tubes in liquid NO2 and store in -80 C
As for the electroporation, I add 1 ul of DNA to 40 ul (10^11 CFU/ml) cells. Set electroporator to 2,5kV and the time constant is usually 5 ms. After the pulse I immediately add 1 ml of ice-cold LB with MgSO4 to the cells (some protocols state that pre-warmed LB/SOB would be better) and recover them at 30 C for 1 hour and then plate on LB + Amp (also, some protocols suggest to pre-warm the plate).
As for the previous steps in Datsenko & Wanner protocol, after many tries i managed to get max 2 colonies for both P. wasabiae + pKD46 and P. wasabiae + pKD3, so it's really not very easy.
I tested the same cells with pBluescript and got a lawn (as expected with high-copy number plasmid), so the cells are atleast a bit competent for electroporation.
If there's any critical info missing in my post, feel free to ask and any help/tips is much appreciated.