I am working with Salmonella and E. coli which contain a mobilizable genomic element. I have a set of deletion mutants, but several times I got very confusing data about plasmid stability using classic replica technique. Plasmid of interest was always wild type in these experiments.
Bow about co transfecting with a reporter plasmid and assying to inform you amount lost rate if thTs what yku wa t to know
Thank you for your answer!
My problem is that detection of the loss of plasmid (selecting and not selecting to antibiotic markers) could be hectic in some cases. We don't know what is behind this phenomenon, but I have made lots of repeated experiments. Conditions were the same in every related experiments. The strain with wild type MGE and wild type plasmid show about 60% loss of plasmid after 8 generations, all the time. Unfortunately the results in MGE deletion mutant strains (made by Datsenko-Wanner one step gene inactivation method) is not so clear.
Suddenly, I thought about two reporter assays as you mentioned above:
1. plasmid-specific ß-gal reporter assay
We can measure the ß-gal activities in the absence and the presence of the plasmid (activity may correlate the population bearing plasmid of interest), but it may be detected hardly because of its low copy number. So, I don't know how reliable this method in this case, but I am going to try it.
2. luciferase reporter assay
It will take some weeks, but I am going to try it. Yes, this could be a solution, if it works. At first, we have to test the microscope in our institute.
Could you recommend another suitable reporter system for me? Cheaper and faster is better. But I am also interested in other quantitative methods. So if you have any ideas, please let me know.
Thank you!
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