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Pectobacterium wasabiae and pCP20 plasmid

electroporation

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6 replies to this topic

#1 anon123

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Posted 17 January 2014 - 08:13 AM

Hello everyone!

 

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.



#2 anon123

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Posted 21 January 2014 - 04:49 AM

A little update to the previous post since I didn't find an edit button. Transformation/Electroporation in P. wasabiae is 2 magnitudes of order lower compared to E. coli (not 2-fold as I mistakenly said in first post), so getting 100 colonies with the latter, it's a very close call with getting any colonies with P. wasabiae. :(

 

The Amp plates I use have the ampicillin concentration of 140 ug/ml. Is it too high or is it irrelevant? I've read that one should use 50 ug/ml plates for low-copy number plasmids and 100 ug/ml plates for high-copy number plasmids. However, since ampicillin does not kill the cells, is it an important factor?

 

I'm really hoping someone has some insight with this problem. rolleyes.gif



#3 phage434

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Posted 21 January 2014 - 08:57 AM

I'm confused. Let me restate what I think you said:

(1) You were able to transform pCP20 into E. coli DH5a chemically with 50-100 transformants

(2) You were able to electro transform P. wasabiae with pKD46 and pKD3 with 2-3 colonies

(3) You get a lawn electro transforming P. wasabiae with pBluescript

(4) You have been unable to transform pCP20 into P. wasabiae

 

Is that right?

 

I would be concerned about (1). You should be able to transform your minipreped plasmid into E. coli with very very high efficiency. This indicates to me that your plasmid prep is poor. What is the concentration of DNA? Have you run it on a gel?



#4 anon123

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Posted 21 January 2014 - 02:14 PM

Thank you for your answer, phage434!

I'm sorry if my posts weren't clear enough, your restatements are all correct.

 

My supervisor got the plasmid from another lab, and it works from them (I don't know which bacteria they use). I did ran them on a gel and here's the result. Sample tagged with '1' is the batch that got me less transformats (lots of RNA in the prep) and '2' is the better batch. I haven't checked the concentration of either of them and I actually ran out of batch nr 2. So, in order to continue my experiment, I should get a new and better prep of pCP20 and I should get a lawn when doing a chemical transformation with pCP20 and E. coli DH5a?

 

Thanks in advance!



#5 phage434

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Posted 21 January 2014 - 06:55 PM

So, yes, transform into E. coli and prepare high concentration plasmid. Then retry your transformation.

You should also try to chemically transform your DH5a E. coli with pBluescript and check that your chemically competent cells are good. The problem with low efficiency on (1) may be bad competence in your chemically competent cells.



#6 anon123

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Posted 22 January 2014 - 02:44 PM

Thank you again for the reply!

 

I'll do another transformation with E. coli DH5a and do a midipreparation to get the plasmid at a higher concentration. The competence of my E. coli DH5a is not really important, since my ultimate goal is to get the plasmid in P. wasabiae.



#7 phage434

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Posted 22 January 2014 - 06:01 PM

Yes, but it would tell you if the problem was a poor plasmid prep or difficulty in transforming into P. wasabiae







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