Hi, I'm using commercial URA3 and LEU2 plasmids from the same serie for plasmid shuffling in yeast. What is actually the risk of recombination between the two plasmids during the URA plasmid chase on 5FOA? As it has already happened to me to isolate 5FOA resistant clones (ura-, leu+) containing the LEU plasmid bearing what had initially been cloned in the URA plasmid... Should I go for fully divergent URA and LEU plasmids in order to minimize the risk? Cheers for the help.
Dear Dominique,
I cannot give you any quantitative information, but yes, this is a phenomenon that we have observed in our group during laboratory evolution of strains that contained multiple plasmids. Very tricky to afterwards reconstruct what has happened ... So, if at all compatible with your experimental goals, try to avoid having multiple related plasmids in a strain, especially during prolonged cultivation.
Good luck,
Jack
Hi Dominique,
The risk of recombination is very high wth Saccharomyces cerevisiae, with almost all strains.
With Alain Nicolas, we have studied this phenomenon some 25 years ago:
Pompon D and Nicolas A (1989) Gene 8(1): 15-24.
Mezard C, Pompon D, and Nicolas A (1992) Cell 71(4): 659-670.
These studies have shown that in S cerevisiae a stretch of 6 bp of identical sequence is sufficient to trigger a high frequency recombination between two plasmids in the same cell.
Best wishes
Philippe
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