It is not clear what you are asking. Could you explain the issue better or the problem you are having?

thanks Dr. Michael. Actually I did cloning and the plasmid was successfully transferred to my bacteria. Now, in case I want to try this in vivo, my engineered bacteria should be free from the plasmids. What is the best way to eliminate those plasmids before I do the test in vivo. Thanks

I presume this is a situation where the plasmid has catalyzed some change in the cell through protein expression or recombination, so that you need cells that had seen the plasmid at some point? Otherwise you could just use the same host before transformation or been transformed with a control plasmid without your gene.

Unfortunately plasmid loss is not so easy to do. The best approach would be to use a plasmid that is designed to be lost, some form of conditional replication for example. There are some plasmids with temperature sensitive origins so you can transform and select at lower temperature where the plasmid would replicate, and then by growing the culture at a higher temperature the plasmid is lost.

It is possible to just look for plasmid segregation and find cells that lost the plasmid. You can grow for a number of generations without any selection (I would 4 or 5 serial dilutions of a culture maybe with a 1000-fold dilution each time to have lots of generations. Then plate out for single colonies and test a lot to identify those that lost the antibiotic resistance of the plasmid. You might need to test a few hundred colonies. High copy cloning plasmids are not lost all that easily unless they have some toxicity associated with them.

Use a plasmid with a marker that can be selected against or use RNA for Cas9 and a gRNA.

Dear Dr Robert, can you clarify more? Or do you have the protocol of it? thanks a Lot.

One of the classic ccounterselection markers is to have the sacB gene on your plasmid and then when you want to select against the plasmid you put on plates containing sucrose.