I am trying to design an experiment to delete LuxS gene from antibiotic resistant E. coli strain CTX-M-15 but I cant use plasmid with Kanamycin because this strain is resistant to Kanamycin, is there any alternative plan to do this kind of gene deletion? I don't want to spend much time on performing such deletion so can someone suggest me an easy and quick method to do that please?
Regardless of which protocol you were willing to follow to produce the desired deletion strain (locus disruption by plasmid integration, I supposed, based on your question), all you have to do is to replace the Kanamycin resistance cassette of your plasmid with another one (e.g. tetR for Tetracyclcine resitance, cat for Chloramphenicol resistance, etc.). This will probably be enough.
Side question. Is the strain resistant to Kanamycin specifically, or to aminoglycosides in general? Depending of the nature of antibiotic resistance of your stain, and the resistance cassette present in your plasmid, you may be able to select the plasmid using Neomycin, Paromomycin, Ribostamycin or GentamycinB. Read https://www.scribd.com/document/24990358/Molecular-Mechanism-of-Drug-Resistance , page 7, for further details. That should be relatively easy to test on liquid culture/plates.
Good luck!
Dear Tanino
Thanks for your answer.. Actually I tested this strain it is ESBL and resistant to many antibiotics like ampicillin, cephalothin, gentamycin, sulphatrid, tetracyclin, and to cotrimoxazole..but sensitive to streptomycin and colistine sulphate..what do you suggest?
It appears you don't have many options available. Selection with streptomycin is possible but not ideal, since E. coli can spontaneously develop resistance (Article Mechanisms of Streptomycin Resistance: Selection of Mutation...
) . Two other selectable options are Chloramphenicol and Bleomycin. Is your strain sensitive to those?
- Badges
- Science topic