I'm having a strange problem with my Agrobacterium GV3101 electrocompetent cells. My master stock of untransformed cells that I used to create competent cells is resistant to rifampicin and gentamycin, and sensitive to ampicillin and kanamycin as they should be. However, when I prepare electrocompetent cells, they somehow take on antibiotic resistance and begin to grow on plates containing ampicillin or kanamycin, making transformation and selection basically impossible. Furthermore, I have tried and had the same problem with two different sets of GV3101 cells (one we had in-house and one donated by a collaborator). What could be the possible mechanism for this? How could I fix it?
Some details:
1. The Agro master stock does not grow on plates containing ampicillin or kanamycin, nor does it grow in liquid cultures containing these (see attached images).
2. Rifampicin is used at 50 ug/ml, gentamycin at 25 ug/ml, kanamycin at 50 ug/ml, and ampicillin at 100 ug/ml.
3. Competent cells are made by streaking the master stock onto a rifampicin plate, growing a single colony in a large culture, and then washing the pellet of cells several times with 10% glycerol and flash freezing in liquid nitrogen. I have tried preparing competent cells by including gentamycin in the plates, but I get the same results.
It's probably just due to the very high concentration of cells in your electrocompetent stock vs. the master stock. I'm pretty sure there's multiple reasons for this. One is that if you put enough cells on a plate, some of them might be blocked from absorbing the antibiotic by nearby cells. Another is that you might spontaneously get some cells that are more resistant, and if you have a ton of cells, the chances of this happening are higher. A third possibility might just be combined power many cells with weak antibiotic resistance/detoxification systems that many species possess. Possibilities one and three seem to be the most likely with the amp plates, because there's essentially no isolated colonies but they're able to grow if the inoculation is heavy enough.
The other possibility is that the preparation of electrocomp cells stresses them out, which causes them to kick on innate antibiotic resistance mechanisms such as efflux pumps.
My suggestions would be:
I have very same problem with C58C1 and ampicillin selection. I am not used to work with Agro and what I see there makes me very curious. First I did single colony from old lab-stock on Rif-Gentamicine and take 24 colonies for amp-resistance check .. more then half were resistant ( I assumed contamination because of students lack of experience) but few were not growing on Amp, I used 200 ug/ml.... So I used that amp-sensitive colony for next step - plasmide transformation .. and whatever I did , chemical or electro , cold or room temperature, it is always the same .. I have plenty of clones in a negative control, just the same as on transformation plates. I rise Amp to 400ug/ml... number of clones reduced a lot compare to 200ug, but no one of 36 colonies had my vector in it, I have checked it with colony PCR and with miniprep.. I can see clear that big helper plasmid , but nothing other then that. ... I really curious about those things. And determined to find the solution to that problem. It cannot be like that, possibly there are some undocumented sub-strains of C58C1 and I am working with wrong one variant. . Meantime I am going to use just another agro strain , LBA4404 that may be better , specifically for AMP selection. I have tried it ones but there was no clones at all even on 50 ug of Amp, so I went for more promising strain that produced lots of clones after transformation ... just to find out that problem - the lack of true vector transformation. Or may be I have those clones but spontaneous resistance background is too high and it is difficult to find them.
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