Hi all,
Could you please explain what is the difference between the replication origin pBR322 and ColE1 ?
If you have look on that vector databases: http://www.embl.de/pepcore/pepcore_services/strains_vectors/vectors/bacterial_expression_vectors/popup_bacterial_expression_vectors/index.html or https://www.embl.de/pepcore/pepcore_services/cloning/choice_vector/ecoli/vectorfeatures, we can see that, for instance, pET-3a is indicated to have a pBR322 origin. However, after SerialCloner analysis, this is the ColE1 origin that is detected.
Is it the same origin? If yes, why some vectors are designated to have ColE1 OR pBR322 in this database ?
Same question with pUC origin. When I look at the pUC57 vectors (for instance), I found a ColE1 origin….
Again, is it possible that several replication origin are on the same vector ? For instance, on pET-28b+, I found ColEI, M13 and F1 ?
Thank you very much for your expertise.
Nt
The short answer to your first question (are they the same ori) is no.
pUC, pBR322, and ColE1 are all close relatives of one another. pUC and pBR322 are both variants of the pMB1 origin. pUC has a few mutations (1 or 2 depending on who you ask) from the pMB1 sequence and these two mutations lead to a 20-35 times difference in copy number - pUC copy number is about 500 per cell while pBR322 is about 20. Plasmids with the ColE1 origin also have about 20 copies per cell (so similar to pBR322).
I don't know the specific differences between the ColE1 and pBR322 origins except that the ColE1 plasmid was named because it encoded the colicin genes from E. coli and pBR322 was named for two members of Herb Boyer's lab. They are different plasmids so their ori sequence is probably different too. Hopefully others can answer this question in more detail
You may want to look at the following publication for some more info: http://jb.asm.org/content/192/14/3654.full.
On to your question about M13 and F1 - those are probably primer sites on the pET28b+ plasmid and not origins of replication. It is very possible to have multiple origins on a plasmid (e.g. a shuttle vector) but you have to make certain that the multiple origins are compatible with each other as each origin type has its own regulation mechanism.
The short answer to your first question (are they the same ori) is no.
pUC, pBR322, and ColE1 are all close relatives of one another. pUC and pBR322 are both variants of the pMB1 origin. pUC has a few mutations (1 or 2 depending on who you ask) from the pMB1 sequence and these two mutations lead to a 20-35 times difference in copy number - pUC copy number is about 500 per cell while pBR322 is about 20. Plasmids with the ColE1 origin also have about 20 copies per cell (so similar to pBR322).
I don't know the specific differences between the ColE1 and pBR322 origins except that the ColE1 plasmid was named because it encoded the colicin genes from E. coli and pBR322 was named for two members of Herb Boyer's lab. They are different plasmids so their ori sequence is probably different too. Hopefully others can answer this question in more detail
You may want to look at the following publication for some more info: http://jb.asm.org/content/192/14/3654.full.
On to your question about M13 and F1 - those are probably primer sites on the pET28b+ plasmid and not origins of replication. It is very possible to have multiple origins on a plasmid (e.g. a shuttle vector) but you have to make certain that the multiple origins are compatible with each other as each origin type has its own regulation mechanism.
Thank you James for the time you spent to answer.
Given that pUC, ColE1 and pBR322 are closely related and are coming from pMB1, we can expect that a plasmid with a pMB1 ori and another one with COLE1 would not be compatible for a coexistence in E. coli, right ?
Nt
Hi
I found this publication that reports on the co-existence of plasmids with same origins of replication.
https://peds.oxfordjournals.org/content/20/7/309.full
I would like to know experts view on this.
Thank you!
That's my paper. We routinely use the fact that plasmids with pUC origins can co-exist in cells for multiple rounds of growth. Although we didn't formally prove it, my sense is that the higher the copy number of a plasmid, the more likely different plasmids containing that origin can co-exist for prolonged periods. Note that this can be a significant problem when creating libraries of any kind, as double (or triple or more) transformation is common, and all plasmids will co-persist, unless any of them is toxic or reduced growth. we examined that in this paper: Goldsmith, M. et al. Avoiding and controlling double transformation artifacts, Protein Engineering Design & Selection 20, 315, (2007).
If you co-transform with incompatible plasmids, as long as they are multi-copy, you can maintain both by selecting for the marker on both, so co-transformation can work. Bacteria will tend to lose one or the other over many generations (this is not instantaneous, and may even take a long time for high copy number plasmids), but continued or periodic selection for both plasmids will kill any bacteria that lose one or the other.
More information here: https://academic.oup.com/peds/article/20/7/309/1546372/Plasmid-incompatibility-more-compatible-than
https://academic.oup.com/peds/article/20/7/309/1546372/Plasmid-incompatibility-more-compatible-than
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