No Colonies after Electroporation of P.aeruginosa - (Sep/20/2013 )
Hi I am following some modified protocols on electroporating P.aeruginosa (PA14) with a recombinant plasmid about 4kb in length but I get absolutely no colonies. I've written down the protocol below if anyone can give me some tips or insights I'd very much appreciate it:
1. Fresh plate of PA14 from frozen stock
2. O/N incubation of LB broth culture
3. 1ml of culture into 100ml of LB
4. inc in 37C shaking inc until OD600=0.5
5. Transfer 20ml to each of two 50ml centrifuge tube (chilled)
6. centrifuge at 5,250g for 10min at 4C
7. Discard supernatant and resuspend in 20ml of chilled SMEB (sucrose magnesium electroporation buffer = 1mM Hepes, 1mM MgCl2, 300mM sucrose, added NaOH to adjust ph to 7, then autoclaved at 121C for 15min)
8. Cent. then resuspend in 10ml SMEB
9. Cent. then resuspend in 400ul of SMEB
10. transfer 40ul to microcent tubes on ice
11. Add 5ul plasmid (about 500ng total dna)
12. transfer to electrocuvette on ice
13. incubate for 20-30min
14. Electroporate at 25uF, 200ohm, and three diff voltages 1.6kv, 2kv, 2.5kv
15. Immed addtion of LB
16. Transfer cuvette content to 15ml centrifuge tube
(there is about 20-30 min of travel time before tubes placed in shaking inc)
17. recovery growth for 1-2 hr at 37C shaking
18. Centrifuge at settings described above and resuspend pellet to about 200ul
19. Plate all 200ul on a single LB Carbenicillin (350 ug/ml) plate
20. Inc at 37C
--> No colonies!
Please help. I am frustrated because the protocols seems to suggest that tons of colonies should pop up each go but I get zilch.
You didn't say what the width of the cuvette gap was. For that voltage, a 2 mm gap would probably be appropriate, but if you used a 4 mm gap, you would likely get no transformants. A 1 mm gap might kill all your cells. It is volts/cm that is the critical number, and it is different for different gap widths. What was the time constant? About 5 ms would be a good number.
The gap is 2mm and the time constant is usually around 5ms. The total 45ul of content in the cuvette fill up about 2mm from the bottom - is that okay in your opinion or would adding maybe 100ul of content to the cuvette more likely to improve results?
I would suggest that you test your cells for viability. Do a transformation as you describe, but plate out on a plain LB plate instead of a Carb plate. You should get a lawn. If not, you are killing your cells somewhere. You can then figure out where. Are you certain your plasmid is correct? A positive control plasmid would be very helpful. You might try this with E. coli instead of Pseudomonas.
I think your incubation on ice in the cuvette should not be necessary, but it probably does no harm. I've not seen Mg++ in electroporation buffers before, but again, I see no particular harm. I would recover with SOC rather than LB. You don't say how much LB you are adding, but more is probably better. The volume in the cuvette is not important, assuming that both electrodes are contacting the liquid.
Are you certain your antibiotic is correct? Where does this plasmid come from? Can you transform E. coli with it?
It's possible there is some weird restriction issue, but this usually just affects efficiency, not eliminating all transformants. I assume PA14 is a "normal" Pseudomonas lab strain.
The cells are not all killed because eventually I observe growth on the antibiotic plates after a few days (also I have performed positive controls - no plasmid, electroporated, plated on LB - resulting in lawn growth that I assume is the same result of plating with-plasmid electroporation broth on LB)
The plasmid I have isolated from Ecoli (I initially transformed the recomb plasmid to ecoli to make a plasmid stock then miniprepped for the plasmid which I used for electroporation) to which it confers antibiotic resistence up to the Cb350 concentration that I use for plating the electroporated cells. The literature uses Cb200 plates (I've seen up to Cb300) but I use Cb350 because it gives me time to see growth on experimental plates before growth starts to appear on the neg control and also the transformed ecoli shows that it can resist antibiotics at the concentration.
I've read chilled cuvettes in most protocols so I just go with that and I have tried simply sucrose (without Mg++) and still get no transformants. I add 1ml of LB
And PA14 is a strain of PA isolated from the clinical setting and there have been studies done using this strain so I am assuming it is a common lab strain or than PAO1
If nothing else comes to mind I can try the SOC medium for recovery and maybe plate on lower concentration plates.
Other than that I guess my big question is (if you have had experience with electroporation) if one normally gets transformants by following standard protocol or do these instances of absolutely no colonies arise frequently/occasionally?
Thanks for your insights,
I'd suggest substituting E.coli for Pseudomonas in a similar electroporation experiment. Same protocol, same plasmid (perhaps a 100 ug/ml carb plate). See if the electroporation ever works. You should get a billion or so transformants. If not, then something is wrong. Try unplugging the cuvette carrier and pulsing. Does it do the same thing? Maybe it's unplugged or has a bad connection.
I found a suitable protocol for electrocompetent ecoli so I will try that along with PA14 as a control...
Also I learned from the protocol that the OD of the culture should not exceed 0.4 at OD600...I knew OD was important but I didn't know that 0.4 was the ceiling (I thought 0.2-0.3 was too low; but then again I saw a bioforum post of another person asking about electroporation in PA14 and s/he used a culture at OD 0.9...http://www.protocol-online.org/biology-forums-2/posts/12606more2.html). So I will try a lower OD value for the starting culture.
My last attempt came up totally blank so something needs to change!