Dear all,
in my lab we are working with E. coli. A couple of weeks ago we tried to remove a low-copy plasmid (pACYC) from one of our strains by simply growing it in the absence of the selective antibiotic. We were also able to confirm loss of the plasmid by both streaking the culture on a plate containing the antibiotic (no growth, as expected) and mini-prep (no plasmid, as expected).
Now, we tried to do the same with the same strain, several times, but apparently it doesn't work anymore. The strain keeps growing on antibiotic. We also checked for the presence of the plasmid, which was positive, to rule out that the resistance got integrated into the genome.
Do you have any idea/explanation why this happens? Have you experienced anything like this before? Is there an alternative technical solution to remove a low-copy plasmid.
You can try the following:
1. Keep doing serial dilution until you get rid of the low copy plasmids;
2. Electroporation - the electric field will induce membrane changes for the plasmids to come out;
3. You can also purify the BAC from your bacterium and transform another cell that does not the resistant gene marker similar to the low copy plasmid; or
4. Plasmid isolation and purification by gel extraction if you know the sizes of your high copy and low copy plasmids
Thanks a lot for your feedback, Alemu.
Just this morning we observed that the plasmid got lost in one of four cases when starting from single colonies. Before we inoculated from a glycerol stock, which may lead to an mixed population of cell with and without the plasmid.
Anyway, important is that it works. Still your suggestions are highly appreciated.
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