i have rosetta game DE3 cells which has chloramphenicol resistance marker gene, it was transformed with one plasmid having ampicillin resistance marker gene and then i made competent cells out of these transformed cells.
now i wanted to transform it with one more plasmid having kanamycin resistance marker gene, i was not able to get any single transformed bacterial colony on Amp-Chl-Kan LB-Agar plate.
is it related to number of antibiotic resistance marker or number/size of the plasmid which can be retained by E. coli cells?
Hey, I have multiple strains of E. coli that carry 3 plasmids with 3 different antibiotic markers. So, there is no problem in using 3 or even 4 different antibiotics for selection. The two most important factors for successful transformations with multiple plasmids are:
1. The replicons have to be compatible. Some plasmids can not be transformed together (http://openwetware.org/wiki/Escherichia_coli/Vectors#Replicon_compatibility)
2. The plasmids should not have any leaky expression of toxic proteins.
Hope that helps. Good luck!
Hey, I have multiple strains of E. coli that carry 3 plasmids with 3 different antibiotic markers. So, there is no problem in using 3 or even 4 different antibiotics for selection. The two most important factors for successful transformations with multiple plasmids are:
1. The replicons have to be compatible. Some plasmids can not be transformed together (http://openwetware.org/wiki/Escherichia_coli/Vectors#Replicon_compatibility)
2. The plasmids should not have any leaky expression of toxic proteins.
Hope that helps. Good luck!
yes, we can have multiple resistant marker depending on the compatibility and requirement.
Yes, it can. The important thing is you have to make sure that all of three plasmids have different incompatibility (inc) group, so that the plasmid can stably coexist in the same E.coli cells.
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