11 replies to this topic

[rmbio]

Hi all

 

I have been doing cloning and transformation almost since past 10 years, but have never experienced the following. In the lab which I have newly joined, my student is trying make the competent cells. While checking the efficiency by transforming pUC18, she is getting some background contamination. This sometimes looks like a mat growth made up of millions of tiny colonies onto which we get larger E. coli colonies. Few other times we get two type of colonies-larger and smaller; I presume that the larger ones are transformed E. coli. When only water is transformed containing no plasmid, no growth is seen suggesting that the contamination is not being introduced during the transformation experiments and that ampicillin which is being used is fine. Even after switching to new plasmid which was newly procured and untouched, same results were observed. Since negative control (water transformation) is fine, I am really wondering what might be going on wrong!!

 

Has anyone experienced the same thing any time?

 

Thanks

 

NB: I have also tried making the competent cells again (from another strain brought from a different lab) which has given the same results.

Edited by rmbio, 16 May 2015 - 12:10 AM.

[perneseblue]

Was the water transformation control done exactly the same as a plasmid transformation? Did you do both experiments side by side?

 

Perhaps the SOC was contaminated?

 

I know of one bench in my lab that if you do cell plating work there, you will get alot of yeast contamination. It is probably due to the presence of an air vent just above it.

[r.rosati]

My best bet is that the antibiotic isn't working (it's below the optimal concentration), so it stunts, but doesn't block, the growth of untransformed bacteria.

 

If the effects range, then perhaps the stock antibiotic is ok, but your student is adding it while the agar is very hot, and the antibiotic degrades (at varying degrees depending on how hot the agar was at the time). You're using pUC18 so adding ampicillin, and the beta-lactams are very sensitive to heat inactivation.

The recommended temperature for adding ampicillin to the medium is 55°C or below. That's roughly when you can keep your hands on the bottle for more than 30 secs without feeling the urge to take them off (of course you know that by experience you just get the feeling). Just in case, tell your student that being able to hold it for 3 seconds doesn't count

Edited by r.rosati, 16 May 2015 - 07:15 AM.

[rmbio]

Thank you perneseblue and r.rosati for your replies.

perneseblue, yes, both water and plasmid transformations were done EXACTLY in the same manner at the same time.  

r.rosati, the thing is that I am not getting any growth with water transformation. If ampicillin would have gone bad or the concentration is wrong, I should have got growth in water transformation also, isn't it?

[r.rosati]

I see. Well, what you describe is really, really similar to what you'd expect from an insufficient amount of antibiotic, though. And when ampicillin is low, given enough transformed colonies on a plate, they'll produce enough beta-lactamase to reduce antibiotic pressure even further and possibly create a lawn of untransformed colonies. While when the transformed colonies are few in number, then what you see are satellite colonies around them, and no colonies away from them. Does this match what you see? If not, then I concur it must be something else.

But would you humor me and double-check the ampicillin stock (calculations, initial purity etc) and the temperature at which it's added to the medium?

Edited by r.rosati, 19 May 2015 - 05:00 AM.

[phage434]

Another thing to check is how fast these small colonies arise.  As r.rosati suggests, beta lactamase will be excreted by resistant colonies. Ampicillin is bacteriostatic and will not kill bacteria, so growth may be possible of satellite colonies.  Usually this happens after more than a day of growth, not overnight.

[rmbio]

Another thing to check is how fast these small colonies arise.  As r.rosati suggests, beta lactamase will be excreted by resistant colonies. Ampicillin is bacteriostatic and will not kill bacteria, so growth may be possible of satellite colonies.  Usually this happens after more than a day of growth, not overnight.

 

 

I see. Well, what you describe is really, really similar to what you'd expect from an insufficient amount of antibiotic, though. And when ampicillin is low, given enough transformed colonies on a plate, they'll produce enough beta-lactamase to reduce antibiotic pressure even further and possibly create a lawn of untransformed colonies. While when the transformed colonies are few in number, then what you see are satellite colonies around them, and no colonies away from them. Does this match what you see? If not, then I concur it must be something else.

But would you humor me and double-check the ampicillin stock (calculations, initial purity etc) and the temperature at which it's added to the medium?

 

Thanks for your suggestions and sorry for delayed reply.

Yes, I have rechecked the concentration of ampicillin and my students has already tried doubling the concetration which gave the same results. Ampicillin is also being added after the medium has cooled down sufficiently. The contaminating colonies are almost arising at the same time of the bigger transformed E. coli colonies (after overnight incubation). Also, the contaminating colonies are sporotically distributed on the plate; they are not closely associated with the transformed E. coli colonies.

 

Thanks

[rmbio]

Hi all

 

I did following changes while making competent cells (1) autoclaved glassware for longer - 30 min (2) considering that the deionized water unit might be the source of contamination, used deionized water from the other institute (3) fumigated laminar hood, incubator shaker and almost whole of the microbiology lab (4) Started the culture from a new untouched competent cell vial which was commercially procured

 

After all this, instead of gettting rid of problems, they are exagerating now.

For preparation of comp. cells, intially 1 ml LB was added to stock vial of comp cell. If was kept at 37C for few hours and then streaked on LB-agar. Clean colonies were obtained on the next day. One of them was transferred to LB. This cultute is now growing but again with the same THREAD like contamination and even it appears that the growth rate has reduced. We are also handling Lactobacillus acidophillus in the same lab and have obsered with those as well that the growth rate has drastically reduced.

Any insights?

 

Thanks

[pito]

Hi all

 

I have been doing cloning and transformation almost since past 10 years, but have never experienced the following. In the lab which I have newly joined, my student is trying make the competent cells. While checking the efficiency by transforming pUC18, she is getting some background contamination. This sometimes looks like a mat growth made up of millions of tiny colonies onto which we get larger E. coli colonies. Few other times we get two type of colonies-larger and smaller; I presume that the larger ones are transformed E. coli. When only water is transformed containing no plasmid, no growth is seen suggesting that the contamination is not being introduced during the transformation experiments and that ampicillin which is being used is fine. Even after switching to new plasmid which was newly procured and untouched, same results were observed. Since negative control (water transformation) is fine, I am really wondering what might be going on wrong!!

 

Has anyone experienced the same thing any time?

 

Thanks

 

NB: I have also tried making the competent cells again (from another strain brought from a different lab) which has given the same results.

 

Actually the first thing that pops my mind is that the transformation is working just fine and there is nothing wrong.

 

What you see is just too much cells... Your entire plate is full (really full!!!) with transformants, there are so many they can not even grow and after a while (yes after longer incubation) you will se a few bigger colonies popping up, surviving (eating) on other cells below/under them.

 

Do you dilute the samples ? What king of volume do you use to transform (volume of the com cells).

And after transformation: you tried to do a miniprep and see if the plasmid is there...

[Andrea Fortina]

Actually the first thing that pops my mind is that the transformation is working just fine and there is nothing wrong.

 

Is it possible that something wrong with the competent cells? Maybe the cells get contaminated during the process making the competent cells?

[OldCloner]

You’ve already done most of the tests I would suggest. Can you dilute enough to get single contaminant colonies (not a lawn) and can you ID the little suckers (like at least a gram stain and colony morphology)? Does the morphology resemble the Lactobacillus? Maybe there is a microbiology lab that can help.  All you need to know is if they are truly not E.coli, as I suspect they are not. Whatever it is seems to be Amp resistant. Are they resistant to anything else you might have in the lab, like Tetracycline? Then the question is where are they coming from?

 

Could it be some buffer you are using to dilute your plasmids but not your water negative control?

 

Have you tried a different plasmid with the same comp cells? I mean one with a different selective marker?

 

Have you recently opened a new bottle of agar stock? If you incubate the agar alone (no antibiotics) is it sterile? Liquid LB as well?

 

You have my sympathies!