Hello,
I am using a 13kb low copy number thermosensitive plasmid transformed into S. enterica LT2 as part of my experiment; I have gotten to the stage where I have obtained the desired mutants and tried curing the plasmid (by incubating nonselectively ON at 37deg, as opposed to 30deg for plasmid stability) only to find that they kept it just fine. I attempted this with wild-type transformants at the same time, and the plasmid stayed within that culture as well.
As there is no universal or low-specificity reliable protocol for plasmid curing, and other curing methods have not been described for this strain and this plasmid, I am at a loss as to what I should be doing next. I will start learning more about plasmid curing, but in the meantime, I have also posted this as readers might offer their expertise if they desire to do so ^^
I would appreciate any suggestions, ideas, or discussion points :D
All the best,
Calin
Some people use growth at 40-42° to cure temperature sensitive plasmids, because sometimes 37° makes the rep protein unstable but there's still a non-zero chance that some of it will fold correctly and allow replication to occur. Sometimes colonies need to be struck out multiple times before you find a clone that's actually lost the plasmid.
This seems less likely, but depending on what temperature sensitive origin of replication being used in your plasmid is, is that LT2's endogenous plasmid is able to rescue it.
If you weren't having the same issue with your empty vector I would assume it could also be due to integration into the chromosome.
Alexandra Johnson Thank you for the reply, definitely useful info!
After further investigating its OR, pSC101 ori, I found that my plasmid is indeed supposed to be cured at 42deg rather than 37deg (although a previously published protocol claimed 37deg ON as sufficient for curing the same plasmid in E. coli)
I'll try your suggestion (matching the other info I managed to find) and incubate ON at 42deg, that should do the trick; I'll come back with a (hopefully positive) update on Saturday morning; will also transform the plasmid into E. coli dH5α and attempt to cure at both 37deg and 42deg, I'm really curious to see if I can get better results than with LT2
Either way, thanks!
Hi Calin,
You can try also these steps: after grow your transformed cells in liquid medium, wash them 3 times with sterile water and resuspend them in a volume of water, then electroporate them as you are trying to transforme them (without add any DNA). You can try 2 or 3 times electroporate them in the same cubete. After that make dilutions and plate these cells. Later on you can check some colonies if they lost your plasmid. Electroporation like that has worked fine for my bacterial cells lost a specific plasmid. Best wishes.
I am not sure you are using a marker, like an antibiotic or a selective medium on a plate.
Alexandra Johnson I managed to cure it in two steps - liquid culture incubation ON at 42deg still yielded very faint bands for the plasmid, but subsequent streaking and 48h incubation (over the weekend; also at 42deg) gave cultures in which the plasmid has been cured completely. Thank you for your help!
Maxuel Andrade Thank you, that actually sounds really ingenious for stubborn plasmids, certainly worth keeping in mind :D A tad more effort, but I am sure it'd be effective, as you said ^^
Mª Angeles Zorrilla Lopez-Perea My plasmid does indeed contain a kanamycin resistance cassette, but I don't think that would be relevant, as I want to select for the cells that have lost it ;)
I'm glad it worked! I think some species/strains are more stubborn about losing thermosensitive plasmids than others.
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