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Dead E. coli

Dead E. coli - Molecular Biology Techniques

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Old 02-05-2009, 07:04 PM
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Question Dead E. coli



I've had a problem recently with the E. coli cells I've been using. I transformed 7 constructs into FusionBlue competent cells (from Clontech), they grew many colonies (significantly more than my control). I performed a colony PCR 5 days later and re-streaked the colonies, but not only did the colony PCR come up with nothing whatsoever (with 12 colonies taken from each plate), but the re-streaked colonies did not grow!

The plate I used for re-streaking was from the same batch as the ones used initially, so it wasn't a case of mixing up antibiotics. 5 days is not normally enough to kill E. coli cells.
This is only the 2nd time I have used these cells, so wondered if anyone else has any experience with them? (the time before worked perfectly with 8 constructs, positive colonies on the PCR and re-streaks growing fine).

They do seem to grow differently to DH5s. They grow very slowly initially in the 37*C incubator and produce tiny colonies, but when in the fridge these colonies overgrow very quickly; DH5s would grow to half the size in several weeks but these seem to grow very large within 2 or 3 days.

For the colony PCR I picked the largest colonies to avoid the possibility of satellites.

Anyone have any idea what happened to my E.coli? Did they just overgrow and die?

Last edited by niki_nje; 02-05-2009 at 07:21 PM. Reason: Accidently deleted paragraph
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Old 02-09-2009, 12:06 AM
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Default Re: Dead E. coli

What kind of plasmid (general cloning, expression?), and what gene did you transform the cells with?

From the sounds of it, because you didn't give us any useful details (!!!!), you might have leaky expression of something that is inhibiting general cellular processes. If you have an expression plasmid, try transforming into something like BL21(DE3) pLysS or pLysE.
Or, you could try growing the cells in the presence of glucose (but you'll have to buffer the media) or glycerol. These act as preferential carbon sources, so the whole lactose metabolism system is shut down (I am assuming the plasmid use T7 polymerase for gene regulation). See how hard it is for us to give a useful answer without good background details?
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Old 02-09-2009, 12:06 AM
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Default Re: Dead E. coli

What kind of plasmid (general cloning, expression?), and what gene did you transform the cells with?

From the sounds of it, because you didn't give us any useful details (!!!!), you might have leaky expression of something that is inhibiting general cellular processes. If you have an expression plasmid, try transforming into something like BL21(DE3) pLysS or pLysE.
Or, you could try growing the cells in the presence of glucose (but you'll have to buffer the media) or glycerol. These act as preferential carbon sources, so the whole lactose metabolism system is shut down (I am assuming the plasmid use T7 polymerase for gene regulation). See how hard it is for us to give a useful answer without good background details?
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Old 02-09-2009, 11:46 AM
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Default Re: Dead E. coli

It was an n-his pet pector with T7.

I have troubleshooted this with my lab with these same details and they figured from the strange biology that because the E. coli grew so fast they probably threw the plasmid out, as all the ampicillin had probably broken down from the presence of all the large colonies. Therefore I am going to re-transform the plasmid into DH5s.

I have used this plasmid hundreds of times in different cell lines, and therefore assumed it was something to do with the biology of these new FusionBlue cells that was causing the problem rather than the specifics of the plasmid. Most E. coli slow down their growth when put into the fridge whereas this strain did not, the Professor in charge of my group said that he didn't like the sound of the way the cells were growing and therefore we are not going to use them again.

Surely if there was leaky expression of a toxic protein the colonies would not have grown so well in the first place? They almost took over the plates they were on. It was only the re-streaks that did not grow. It would be a bit odd that so many big fat colonies grew, but yet 96 of those from 7 different plates suddenly had leaky expression when restreaked onto the same type of plate.

Last edited by niki_nje; 02-09-2009 at 11:49 AM.
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