24 replies to this topic

[fishdoc]

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

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

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.

[HomeBrew]

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:

Quote

Pseudomonas aeruginosa isolates are highly resistant to chloramphenicol (minimal inhibitory concentration, 100-1,000 ug/ml). Most of the strains tested produce the enzyme chloramphenicol acetyltransferase (CAT) while 15% do not produce the enzyme, although they are [still] highly resistant to the drug.

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.

[green_bear]

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

Edited by green_bear, 29 January 2010 - 07:09 PM.

[fishdoc]

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

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

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.

[christy]

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:

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

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

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.

[phage434]

For electroporation in my organism I buffer the electroporation buffer with 50 mM HEPES at pH 7.5. Sucrose alone will not control the pH, but I don't know if that is an issue with Pseudomonas.

[fishdoc]

christy, on Mar 2 2010, 09:15 PM, said:

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

Without knowing much about the vector, 1.5 ug seems like a lot to be using for transformation. I think, on average, I use between 50 and 150 ng for transformation, but I'm also not working with 11 kb vectors. I don't have a lot of experience with plasmids larger than 8-10 kb, but I think the larger ones are inherently more difficult to get in.

[dryeliz]

green_bear, on Jan 30 2010, 05:49 AM, said:

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

Hey everyon,
I am new at the forum and I have started to study with P.aeruginosa. I want to tranfer a plasmid from E. coli to P. aeruginosa. I am waiting your suggest ?
Bset regards.

dryeliz

[rameshcdri]

Hi Guys, I am new member of this forum. I am working on Pseudomonas aeruginosa.

I went through the topic 'Problem with electro-transformation to Pseudomonas aeruginosa' which really helpful for me.
I electroplate the non-replicative plasmid pEX18apGD by using 10 min protocol. After 2 and half hour recovery incubation, I plated 500ul to Gen selective plate (30ug/ml; without centrifuge). After 24 hr there is no colony appeared but after 48 hr there is very small colonies are appeared. Is this real colonies? or son specific colonies due to breaking down of Gent. antibiotics. The non-selective plate also have same texture of colonies. Can you please suggest me to go further to screening the my gene of interest?

Ram

[rameshcdri]

green_bear, on 15 January 2010 - 02:31 AM, said:

Hi everybody, I am new to this forum. Anyway, I would really appreciate if you could give me some suggestions on this.
Currently, we are trying to get a 3kb plasmid containing RFP constitutively expressed into P.a. We are following the protocol in this paper:
10 minutes preparation of electrocompetent P.a.

I will summarize the steps which we used as below:

Quote

A. Prepare electrocompetent P.a.:
1. Inoculate single colony in 6ml of LB overnight.
2. Distribute cell culture equally into 4 microcentrifuge tubes.
3. Centrifuge at 16000 x g and room temperature for 2min and discard supernatant.
4. Wash each tube of cell pellet with 1ml of sucrose solution and centrifuge. Repeat Step 4 twice.
5. Resuspend all the 4 cell pellets with a combined volume of 100ul of sucrose to produce 10^-9 viable cells.

B. Electroporation:
1. Transfer 3ul or 500ng of purified DNA into 100ul of electrocompetent cells.
3. Transfer the mixture into pre-chilled cuvette.
4. Slot cuvette into BioRad Gene Pulser and pulse shock (2.5kV, 200Ω and 25µF). Add 1ml of LB at room temperature immediately into the cuvette. Transfer mixture into 1.5ml microcentrifuge tube and shake for 1 hr at 37oC.
5. Centrifuge at 16 000 x g and discard 900ul of supernatant. Resuspend cells in 100ul of residual medium.
6. Plate entire cell culture onto LB plate with suitable antibiotic (Chloramphenicol 20ug/ml). Incubate at 37oC overnight.

The only 2 steps which are slightly different from the paper are in bold:
- After the pulse shock, it takes us about 30 seconds (not immediately as suggested) before we add LB, as we need to move the sample into fume hood.
- We incubate the cells for 1 hour in 1.5 ml microcentrifuge tube, not glass tube.

The problem we encounter is that, after this 1-hour incubation step, the cell solution develops a transparent glue-like matrix which attach to all the cell pellets. We suspect that this might be biofilm, but have no idea which leads to its formation. So we always end up plating both the cells and this matrix on the plate. The next morning, we have indistinct colonies growing all over in all the plates, including negative control (cells without plasmids added). So it might be that this biofilm protects the cells from the antibiotics.

Have anybody else encountered this problem before, or had any suggestion on this?

Thank you very much in advance :-)

G_B