I have 3 plasmids; kanamycin, ampicillin and chloramphenicol- as markers and the plasmids are compartible. Can these plasmids be retained in E. coli without becoming a burden or should I transfer some genes to the genome? Any experience?
Make sure you've checked plasmid compatability groups based on the origins of replication (simply checking that different resistance markers are present is not adequate). As long as you maintain selective pressure, all plasmids will be maintained. However, to simplify your system, it may make more sense to put all three transgenes in the same plasmid.
There are now a whole host of cloning methods that allow custom assembly of vectors with multiple gene cassette or regulatory elements, so this is approach is much simpler than it used to be. Unless you are trying to do in vitro evolution where you need to have the genes be under different selection cassettes, a single plasmid approach might be more elegant than the 3 plasmid solution, and simpler than genomic targeting.
Thanks a lot Daniel for your advice. I kindly request if you have some protocol or some reference which i can refer to so that i can do with one or two plasmids.
The protocols I like best for assembling multiple DNA cassettes are Gibson cloning or the SapI strategy published for MultiLabel cassette by Philipp Berger's group (PMID 21081918). Tried and true.
Alternatively, ff you already have cassettes in ENTR vectors, you can use Gateway recombination-based cloning.
Another point of consideration is whether you need to have similar levels of expression. Different plasmids will often have very different copy numbers that will affect expression levels due to gene dosage. This may or may not be a concern to you. But it is possible to have 3 plasmids in a cell. There will be some burden imposed in terms of growth rate, but probably not terribly severe.
- Badges
- Science topic