I am trying to remove pCAMBIA2301 from EHA105. I tried to grow without kanamycin at 37C for several generation. but EHA105 still contains Kan resistance. Is there any alternative or and changes that need to be made?
Have you seen this paper (attached). See the method they used and described in the Result paragraph "Removal of plasmids pLR581 and pLR585 from L. reuteri ATCC 55730". I am wondering that whether this same method can be used for the Agrobacterium.
Paper title: "Removal of Antibiotic Resistance Gene-Carrying Plasmids from Lactobacillus reuteri ATCC 55730 and Characterization of the Resulting Daughter Strain, L. reuteri DSM 17938"
There is a similar discussion on this subject on RG: How to get an E. coli strain free of plasmids?
https://www.researchgate.net/post/How_to_get_an_E_coli_strain_free_of_plasmids
See Dr. Benedik's reply:
Michael J. Benedik · 108.17 · Texas A&M University
The above methods should work fine - given sufficient patience. I have frequently just screened after many generations, although EtBr or MitC does seem to increase the frequency (but has some increased rate of mutation going along with it). If your plasmid turns out to be very difficult to cure, you might find an oriR6K plasmid with a different and counterselectable marker on it, then you can displace the resident plasmid with selection and evict the new plasmid with counterselection.
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