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Plasmid curing method


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#1 Qundo12

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Posted 27 October 2009 - 03:49 AM

Recently I isolated a bacterium with some good properties and I wanna engineer it. However, this bacterium has so many native plasmids (4 or 5), which is not good for maintaining a transformed plasmid. I want to eliminate them and I tried Ethidium Bromide. However, after some rounds treating the cells with sublethal concentration of EtBr, its plasmids were still there. Is there anybody has experience with plasmid curing? is it possible to use another convenient method instead of EtBr? Thank you for every comment and suggestion.

#2 Julio-Claudian

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Posted 27 October 2009 - 06:48 AM

Those guys in my lab working on wild type Klebsiella pneumoniae did it by subculturing the organism many times. Lots of plates (non-selective) to grow them and also selective plates to check if the antibiotic resistance (hence plasmid) still present.

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.


#3 fishdoc

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Posted 27 October 2009 - 07:34 AM

I've never used it, but I know that growth on acridine orange has been used to cure plasmids. It might be worth some effort to see if any related organisms carry similarly sized plasmids, or if the sequence of these plasmids is known. No use in trying to cure a plasmid if you can first determine whether or not there are plasmid addiction genes encoded by them.

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.

#4 HomeBrew

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Posted 27 October 2009 - 12:43 PM

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...

#5 Qundo12

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Posted 28 October 2009 - 03:02 AM

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...

Thank you all for suggestions ^^!
@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 !!!!




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