Competent cells not growing - (Nov/13/2007 )

Hey guys,

Our competent cells (SURE cells from Stratagene) haven't been growing well in culture recently. Sometimes they grow well but other times (more and more) they don't grow at all. We're not exactly sure why this is happening, the clones are definitely there, we think it's the cells themselves,. To confirm this my boss performed two transfections of the same empty vector into two different strains - XL10-Gold (Statagene) and SURE cells. The XL10-Gold cells grew well but the SURE cells didn't grow at all. We make our own competent cells and this batch of SURE cells has worked well before but has not been working well (or consistently) recently. I was just wondering if any body knows more about this type of thing. Do competent cells go off? How would that occur?

Cheers, Rob

You might check that they are still tet (and kan) resistant. Tet resistance is carried on an F plasmid, which might be lost. It also carries proAB, which (missing) could cause a growth defect. You could also think about phage contamination. XL10 Gold also has a similar F plasmid, but perhaps it has not been lost, or is more stable for some reason.

Cheers for the post Phage, I appreciate your knowledge on this subject because I don't know a lot about E. coli genetics and would like to learn more. It's actually quite ironic that you said what you said. I just made a streak plate of new SURE cells today (using a new Stratagene vial) on a kanamycin plate, the only reason I didn't make it on a kanamycin/tetracycline plate was because I didn't have any tetracycline.

Back to the faulty bacteria though, my boss was just saying today that the bacteria might have lost some genes. How would you test for the loss of the F` episome? You couldn't do it by antibiotic selection because the cells are not growing under any conditions any way. Could you do a PCR for one of the genes on the episome?

Although your suggestions were interesting I'm still a little sceptical. The cells have slowly begun to stop working, which makes me feel like it's a degradation-type thing that's occured over time in the -80C freezer. Is loss of the F` episome going to happen in such cold storage conditions? And can the phage contamination be as sporadic as the growth of my cultures have been?

I've just been reading up on the ProA and B genes and others and phage contamination testing - fascinating.

Cheers for the post,
Rob

I'm skeptical of this thought too, but it is a cheap experiment. If you have truly lost the F plasmid, then the cells will no longer be tet resistant, since that resistance gene is carried on F. That won't be true of Kan, since it is chromosomally located. Given that you are going back to the (presumably correct) new vial from Stratagene, this problem (if present) would go away.

Yeah, the new cells should be fine. I'm more interested than anything why the old ones stopped working, it will be good to know why so I can avoid it or at least test for it again if it happens again. I was thinking about the tet resistance on the F` episome but I can't select for it because the cells aren't growing. Even in plain LB they won't grow, so I can't use growth as a test.

Just as an update, even the new cells I got from the original Stratagene vials did not grow - but I think these cells have been there are while, although they have never been out of the -80C. Maybe the cells just get old after a while. I'm still sceptical, it seems strange that our own made cells and the original cells have both stopped working at the same time, although maybe time is the explanantion. We've ordered some new SURE 2 cells, they'll be fine.