Plasmid curing method
Posted 27 October 2009 - 03:49 AM
Posted 27 October 2009 - 06:48 AM
They seem to get it and now moving on with their work.
Edit: Copy number influences the plasmid stability so you may need to do more, longer. Cheers!
Edited by dreamchaser_jc, 27 October 2009 - 07:10 AM.
Posted 27 October 2009 - 07:34 AM
I work with an organism that has 2 plasmids, and there were many attempts done using common curing methods, and they weren't able to cure the plasmids. They could drive the copy number down so that the plasmids were not detectable in a miniprep, but when removing the curing pressure, the plasmids came right back. We've since been able to use another plasmid, pBBR1, which is not incompatible with the native plasmids. It does not affect virulence or any other process that we know.
Posted 27 October 2009 - 12:43 PM
Posted 28 October 2009 - 03:02 AM
Thank you all for suggestions ^^!
I too have heard of acridine orange being used for this purpose. I've also heard you can use electroporation to cure plasmids -- see, for example, here, as well as other chemicals (novobiocin, SDS), or by growth and passage at high temperature. There are also apparently commercial kits for this purpose -- see here, for example...
@dreamchaser_jc: yes, it's possible, maybe some copies were deleted but the others are still remaining. I just did 2 rounds of treating with EtBr and the Kanamycin resistance of this strain reduced, but the confirmation by plasmid extraction revealed the presence of many plasmids.
@fishdoc: actually, I read a paper comparing the effects of acridine orange and EtBr on plasmid curing and they conclude EtBr is much better, so I chose this one for my experiment. This is the newly isolated strain in my lab, so there is no information about the native plasmids, but I guess they confer the antibiotic resistance. Moreover, when I use a vector from a related strain (same family), it's risky that the vector is incompatible with the native ones (I failed many times with electroporation).
@Homebrew: the paper you introduced about eletroporation for plasmid curing is very interesting. The idea is quite simple but efficient, I guess. The poration on cell membrane is good for plasmid entering the cell, but I haven't realized that it's also good for plasmid escaping. I'm going to try this method, plus ErBr treatment with more rounds, maybe this could help.
Thank you guys again !!!!