Electroporation of stem cells with DNA in Top10 - endotoxins? - (Sep/17/2006 )
The aim of my work is to produce transgenic mice by targeted mutagenesis of ES-cells. Therefore I grew the constructs I wanted to electroporate with in XL1-blue bacteria and isolated the plasmid with the GenElute™ Plasmid Miniprep Kit from Sigma and purified it with phenol/chloroform. This method is well established in our lab and the electroporations usually work.
But now I have got a construct which doesn't grow well in XL1-blue, but in Top10. I again isolated that new construct with the GenElute™ Plasmid Miniprep Kit, purified it and did the electroporation. Bur after that all my ES-cells were dying and no clone at all survived. I repeated the experiment, but this time I used an endotoxine-free purifying kit from Qiagen. The electroporations worked again.
This led to the conclusion that Top10 bacteria may produce a for ES-cells lethal amount of endotoxins.
My question is now: Is something known about the amount of endotoxins that is set free by different E. coli strains? Are there special references about Top10 or XL1-blue bacteria and endotoxins?
Thanks a lot for your help!
well, another problem is that endotoxin in ubiquitous in the environment. so, even if your e coli strain produces less, then you are still getting some from the water, media, buffer, etc. even double distilled deionized autoclaved water will carry some endotoxin. it is possible to get stuff that is endotoxin-free for the experiment, although it is more expensive
first, thanks a lot for your answer!
I think youre right, perhaps the endotoxin-free stuff could increase the efficiency of the electroporation. but as I said, the experiment fortunately already worked :-)
my problem is now to get some information about xl1-blue and top10, because I have to discuss this endotoxin problem... is there anything known?
I don't know, but I suspect you may only be able to answer that question via Pubmed and a literature mining expedition
the way I understand it, based on tissue culture treatments in my lab, it doesn't take much endotoxin and if you're getting significant carryover with your plasmid from either strain, it will be a problem. I don't know that you can expect one to be significantly worse than the other? I guess I am surprised that you haven't experienced endotoxin toxicity before with your electroporations?
A Gram-negative bacterium contains approximately 10-15 g LPS; i.e. 1 EU can be generated by only 10^5 bacteria.
Escherichia coli contain approximately 2 x 10^7 phospholipid molecules per cell, whereas each cell contains approximately 2 x 10^6 LPS molecules (1).
LPS comprises 3% of the total cell dry weight in typical laboratory strains of Escherichia coli such as E. coli K-12 and more than 10% of the total cell dry weight in smooth, wild-type E. coli isolated from clinical sources (2).
1) Raetz, C.R. (1986). Molecular genetics of membrane phospholipids synthesis. Annu. Rev. Genet. 20:253-295.
2) Neidhardt, F.C. Chemical composition of Escherichia coli. In: Neidhardt FC, Ingraham JL, Low Jr KB, Magasanik B, Schaechter M, Umbarger HE, eds. (1987) Escherichia coli and Salmonella typhimurium, Cellular and Molecular Biology. 1st ed. Washington, DC: American Society for Microbiology, 3-6.
More information regarding endotoxins you find on: www.endotrap.de