Two same oris (origins of replications) in one plasmid - is it possible? (Oct/23/2005 )

Hello everyone!

I need your help.

If I have the same two e.Coli oris in one plasmid, will e.Coli able to replicate plasmid like this? (high copy)

It will be very helpful if you answer a.s.a.p

Thanks
L.k

---

hi
i think it's impossible to get proper replication of plasmid. it possibly may be eventuall possible if the Ori are in the same direction on the plasmid. But i really don't know. But if they are facing,i think it's impossible. (when two ori arer putted in a bacterialchromosome it's lethal if i'm not wrong...)

---

Thank you Fred.

I am happy to hear your answer. If you have some reference regarding this issue I will be very grateful

Bye

---

I have no references, but just going on my intuition and experience, I agree with Fred -- I doubt very much this would be a stable plasmid. I suppose given some time and selection, you could isolate a mutant in which the two origins get along (i.e. one has mutated), but you would likely need to first integrate it into a host chromosome, if it doesn't natively replicate at all.

Let's back up a minute, though. Why do you want to do this?

---

Hi!

I wanted to get one big plasmid (~10kbp), with luc gene, so I have decided to put together two plasmids, without deleting any sequence. But, I havn't got any recombinants. So, I need an explanation why is that. I suppose it's because the ori's, but I have seen an article which made a plasmid with two/three ColiE oris, and they show that if it's in the same direction so it's maybe Ok, but if they are not, so one of the ori becomes " a polar ...pause" ( I am not familiar with this expression).I have not seen any other articles on this subject. I would like to know more.

Thanks a lot anyway:)

---