Plasmid stability over a 100%? - Why does ampicillin increase cell viability? (Mar/31/2007 )
Hi, I'm a Spanish student in 3rd year of biotechnology.
We did a plasmid stability test of a pET21d (into which we had cloned the DHFR gene) in E. Coli BL21DE3pLys. For some reason the number of colonies obtained in the ampicillin+ plate was larger than the plate with no additives (around a 130%). It could be a coincidence but all the groups obtained similar results.
There might be something obvious that I'm not taking into account, but does anyone have an idea or a clue of why this might be happening?
It seems to be some simple mistakes made somehow in the experimental process. Have you repeated the experiment to see if this is repeatable? Its hard to be the detective here.
But how many colonies more ?
and, well, is pET21d contain only Amp resistance gene?
Thanks for answering and sorry I took so long to reply, I was out of town.
Since this is directed lab experiment we don't have the chance to repeat it ourselves but apparently this has been happening ever since they started doing this experiment a few years ago, which isn't that surprising considering we've all been using the same cells (so it's got something to do with the strain).
Since the transformation of these cells was done by other people I will never know exactly what happened but I'm trying to come up with ideas that could explain why they grow either the same (in some cases) or more (in our case) in the presence of Amp.
As for how many colonies more, as I've said, not all the groups obtained more colonies in Amp but it was a frequent outcome. I've checked the numbers again and it's not an obvious result, for instance, in a 10-7 dilution we obtained 68 colonies in LB and 96 in LB + Amp.
I'm beginning to think it's just a statistical error, and it only means that it's a very stable plasmid, but it's surprising that a lot of us are coming up with the same results... what do you think?
As far as I know, pET21d only has the Amp resistance, but I will check.
It has probably something to do with pipetting errors when doing the dilutions. Maybe the pipettes are just miscalibrated. Or the ampicilin might be degraded... Nevertheless, it doesn't really matter if those colonies have the plasmid of interest.
Other than that, the BL21 strain isn't really very stable for the plasmids. Here in the lab we use them for protein overexpression, but do the transformation everytime instead of keeping a stock of those transformants. I suppose they are very prone to mutations or something.