Hi. I'd like to insert two plasmids to E. coli strain, but
1. I wonder whether they are compatible. One plasmid is pTrc99A of which the origin is pBR322. And the other one is pASK-IBA3 plus, of which the origin is colE1 and f1.
2. Actually, these plasmids both have AMP resistance. Would it be possible that one plasmid will be lost since one is enough to allow the cells to grow in the presence of ampicillin?
Thank you very much!
Since f1 is a single-stranded bacteriophage and pBR322 is a double plasmid with a colE1 replication origin, I would be very surprised if they were incompatible.
Hi Thanks for the answers. So I just double checked with the company and found that actually the origin of pASK-IBA3 plus is ColEI. It has both ColEI and f1. Now would it still be compatible?
Referring to my attached link, it is recommended that plasmid carrying ColE1 and plasmid carrying pBR322 should not be co-transformed. Now I am pretty confused
http://blog.addgene.org/plasmid-101-origin-of-replication
These plasmids are not compatible. It may be possible to maintain them together in the same cell if you maintain selection for both plasmids, but as soon as the selection is released they will start producing segregants with only one plasmid type. Even with selection, the copy number of each plasmid will fluctuate wildly.
ColE1 and pBR322 have essentially the same origin and thus are incompatible. As above you can maintain both plasmids in a culture given different selectable markers but there will be cell-to cell variation in expression levels. Also the plasmids could recombine to give a single large plasmid.
Hi Andrew and John, thank you so much for the help! I think I will just incorporate the two protein of interest into one plasmid.
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