Problem with electro-transformation to Pseudomonas aeruginosa - (Jan/15/2010 )

So, if the transformation protocol is under control, you might want to check a few other things. A P. aeruginosa restriction system could be cutting your DNA. You might want to check what restriction systems are typically present in your strains. Do you have a control plasmid that is functional in Pseudomonas that you could use to verify transformation? What origin of replication are you using in your plasmid? Have you sequenced your plasmid to assure that it is what you think it is?

When I worked with P. aeruginosa, we usually moved plasmids in via conjugation (see Goldberg and Ohman. (1984). J. of Bacteriol., 158:115-1121), but your vector must be mobilizable by a helper plasmid. Recently, I successfully transformed PAO1 using cells made competent by the rubidium chloride method, so that works, too. But you need to get your selection method tightened up so that you have no growth at all on your control plates, or you're going to have difficulties with any method.

Hi guys, thanks so much for your replies!

@fishdoc: Yes, I meant beginning of a thin lawn.

fishdoc and phage434, you are both right, I should check with the person I got this plasmid from about the its compatibility with P.aeruginosa. But say, I know the sequence of my plasmid, is there any way I can do the check myself online? I also have the RFP insert in pUC19, a common E.coli vector. Does anyone know whether this vector is compatible in P.aeruginosa? There's also the 12kb RSF1010-derived plasmid which is P.a.-compatible, but the last time I tried extracting the plasmid, I could not see any band under gel electrophoresis.

As Homebrew mentioned, I am still not 100% sure about the selection method. Last time I tried streaking colonies directly from a P.aeruginosa plate into LB plates of 20,30 and 50 ug/ml Chloramphenicol. Cells did not grow in the first plate, while I could see lawns on the other two. But the fact that untransformed cells did grow when 200ul of recovery culture was plated (and even 50ul after 24h) makes me a bit doubtful of selection condition. I am not sure whether this is due to, as fishdoc suggested, "the mass of cells plated overcomes the antibiotics present in the media".

In addition, which OD of cells should I use to prepare electro-competent cells? I notice that the protocol mentioned overnight culture in LB was used. But this is kinda weird, cause after overnight incubation, normally the bacteria already reached stationary phase and might not be that active any more...Do you guys think I should re-inoculate this culture in the morning (about 100 times dilution), and grow into an OD of 0.5-0.7?

And any comment on the washing and electroporation medium? I used 300mM sucrose (dissolved in distilled water) for all the steps.


G_B

I don't have the protocol in front of me, but we make E. coli electrocompetent by growing an overnight culture, then using that to inoculate about 40 mls of LB (1:100 inoculum) and growing for a few hours to an OD of between 0.6 and 1.0. Cells (10 mls) are put into centrifuge tube and pelleted, resuspended in DI water, pelleted, resuspended in DI water, pelleted, resuspended in 10% glycerol, pelleted, resuspended in 2 ml 10% glycerol, transferred to 1.5 ml tubes, pelleted, resuspended in 200 ul 10% glycerol, put in 1.5 ml tubes (50-80 ul) and stored at -80C.

Hi, just a quick update from today.
I stored recovery culture left over from last time electroporation in 4degC fridge. Yesterday I took 100ul of it to plate on LBC plates (30ug/ml). After 16 hours, I saw some colonies on the plate, but none on negative control plate. But as the colonies were still growing (I could not pick them from the plate cause they were not tightly attached to the agar surface), I let it incubated further more. About 1 hour later, I saw lawn of bacteria on both plates. So is this due to break-down of antibiotics?

G_B

green_bear on Jan 28 2010, 11:02 PM said:

Without being there to see it and with no past experience, I can't say for sure. Only that it's possible, but there may be other possibilities as well. On occasion, in electrotransformations or conjugations, the plates end up with some colonies that grow early, but eventually a small lawn grows up. But because those early colonies started growing, they stand out from the lawn as they are much larger by the time the lawn is there. So you can pick from the center of those larger colonies and transfer to another plate, then screen for the correct construct. If you haven't already, you may want to take a sample of your P. aeruginosa electrocompetent cells and streak for isolation to make sure you don't have a contaminant in there.

As for the colonies not being tightly attached, does that really matter? You should still be able to pick that colony with a toothpick or pipet tip and transfer it to another plate to let it grow up and screen. Once you've taken that sample off, you really don't need that original plate anymore, presuming the patch grows.

Chloramphenicol is inactivated by a chloramphenicol acetyltransferase, which I believe is a cytoplasmic enzyme, and is not secreted. Therefore, it's hard to imagine what you're seeing is the result of destruction of the antibiotic in the surrounding media, as one would expect with bugs producing beta-lactamases, which are secreted, and which physically destroy the antibiotic in the media, allowing satellite colonies to arise.

It's more likely that your recipient strain is resistant to chloramphenicol. According to this abstract:

I think you either need to really jack up the concentration of chloramphenicol, or find another vector that will allow you to select your transformants by a different method.

Hey guys,

I finally successfully transformed the plasmid into P.aeruginosa!!! This morning I found a plate of as many as 100 red colonies, and none in the negative control plate.
I think the key problem here is the OD of sample which I used to prepare electrocompetent cells. This time I used those with OD 0.9. LBC plate of 30ug/ml, 100ul out of 1ml of recovery culture was plated.
@Homebrew: thankfully, the strain which I am dealing with now is very sensitive to antibiotics, so I guess chloramphenicol did a very good job!

The best thanks to Homebrew, fishdoc, and phage434 for all your helps throughout this problem!

Best regards, and have a nice weekend!

G_B


P/S: Could I just store this plate in 4degC fridge? Or what is the best way to keep it? I'm gonna repeat this protocol one more type with a similar plasmid. So until I confirm its efficiency, the plate is invaluable to me :-D

green_bear on Jan 29 2010, 08:49 PM said:

If the strain is that important, grow up some overnight cultures and freeze at -80C for optimal security. By most accounts, 4C is fine for a day or two.

Personally, if I'm working with a mutant or transformed strain (I work with a pathogen), to reduce any sort of risk of random mutation that could result in a virulence defect, I always use the minimal number of passes and get the strains in the freezer as fast as possible.

Congrats on the success.

Hi Fish doc,

I am working with Pseudomonas denitrificans, I was struggling with pUC based plasmid transformation, my plasmid size is 11 Kb. Finally, i used 0.8 OD cells and incubte at 40 deg for 20 with 1 U/ml alginate lyase to degrade the alginate, after harvesting the cells, I used 300 mM of sucrose as transformation buffer, 1. 5 microgram DNA, regeneration for 1 hour and 30 mins and transformed by electroporation 2.5 kV, but i got only one transfomant. Any ways, i suceed.
Now, i am in need to transform 12.5 Kb plasmid in wild type P. denitrificans. Now, i couldnot get any transformant. Can u give me some suggestions. it will be very useful for my work.

Thanks in advance,
Christy

fishdoc on Jan 30 2010, 01:48 PM said: