Hi all, I would like to know whether the coexistence of two plasmids can lead to a low plasmid copy number of each of them? Assuming that these plasmids have a compatible origin of replication. We had a problem where we coexpressed two plasmids, but we couldn't see the over expression of one of the target proteins, is this perhaps a rational explanation?
If anyone can help or suggest a paper to read about this I would appreciate. Cheers!
Do you know the origins of replication on your plasmids? If so, you can check for compatibility. What are the promoters/plasmids driving expression of each of your transgenes?
In both target proteins is pMV261 which allows for constitutive expression under the control of the Hsp60 promoter
Technically, I'm having trouble understanding this experiment. If you want to express two proteins, better to make a bicistronic message under the control of a single promoter. If you want to use two plasmids, you'd need to have two different vectors, which rely on orthogonal antibiotic selection methods, and don't fall into an incompatability group for the origins of replication.
If you clone two different genes into one vector, and try to co-transfect these two versions of same plasmid, you will have no guarantee that transformants harbor both variants of the plasmid. Moreover, as the bacteria grow and divide, you'll have no way to maintain a balance of the two plasmids in the progeny (if you were lucky enough to get a high rate of co-transfection initially), and over time, you'd end up with cells harboring just one of the two transgenes. It sounds like this is what you're observing. If I misunderstood your experimental design, please explain it in more detail.
Hi Daniel,
We were using two different plasmids (one gene each) with orthogonal antibiotic resistance: kanamycin and hygromycin, of course with the same origin of replication for Mycobacteria. While we could prove over expression of one of the proteins, the other one, which was a GFP, showed basal levels of expression. Perhaps the cells weren't simply happy with the other one and ejected it? Or spending more energy on expressing one but not the other?
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