A question about pBR322 and pUC ori - (Mar/06/2011 )
I have a question regarding the compatibility of pBR322 ori in pET vector system and pUC ori in pUC19. As far as I know, they are basically the same except one mutation and the absence of rop gene in pUC19 so it has more copy number. In E. coli, if I want to maintain the 2 vectors in one cell with middle and high copy number, seems like only these ories can help. Actually I was suggested by one of my senior to do that and he also did the same thing. I'm going to construct a pET derivative vector with Amp resistance and a pUC - pACYC hybrid vector (with p15A ori replaced by pUC ori fragment) with Kan resistance. My senior used pET and a TOPO vector with both Amp and Km resistance and seems like they worked.
So, I wonder that the copy number of the pUC derivative vector would be reduced or not, since the rop gene presents on pET vector. The second thing is maybe this system will not stable, especially when I use BL21 as the host (rec+ strain), these ories can be exchanged.
Is there anyone here has experience with this kind of two plasmid system or can help me to make it clear? If you have any better suggestion for the system, you're very welcome, I'll very appreciate that.
in principle it should be possible to maintaine both plasmids stable when selection pressure is used ...i must confess i do not fell really cosy with the idea using two plasmids with the same ori and therefore i avoid it when possible (and most of the time it is since we have several origins from which we can choose.
You can think about replacing the pET for a pETcoco™ ...see the corresponding info here.
Regards,
p
Why do you need two plasmids with different copy numbers?
Quasimondo on Sun Mar 6 16:06:25 2011 said:
I have a question regarding the compatibility of pBR322 ori in pET vector system and pUC ori in pUC19. As far as I know, they are basically the same except one mutation and the absence of rop gene in pUC19 so it has more copy number. In E. coli, if I want to maintain the 2 vectors in one cell with middle and high copy number, seems like only these ories can help. Actually I was suggested by one of my senior to do that and he also did the same thing. I'm going to construct a pET derivative vector with Amp resistance and a pUC - pACYC hybrid vector (with p15A ori replaced by pUC ori fragment) with Kan resistance. My senior used pET and a TOPO vector with both Amp and Km resistance and seems like they worked.
So, I wonder that the copy number of the pUC derivative vector would be reduced or not, since the rop gene presents on pET vector. The second thing is maybe this system will not stable, especially when I use BL21 as the host (rec+ strain), these ories can be exchanged.
Is there anyone here has experience with this kind of two plasmid system or can help me to make it clear? If you have any better suggestion for the system, you're very welcome, I'll very appreciate that.
Thank you so much for this information, I didn't know about this pETcoco™ system before. I'll discuss with my colleagues to decide ordering this plasmid or not. In our lab we're developing a kind of system that the proteins encoded by the first plasmid will bind to the second plasmid so we need to couple 2 plasmids with different copy numbers to evaluate this system. In my hand now, I just have p15A, pSC101, pUC and pBR322 ori, so pUC and pBR322 is my only choice. Actually right after posting the question here I found out an interesting paper in which they concluded that even the 2 plasmids utilizing the same ori, they can be maintained under the pressure of the 2 antibiotics: Plasmid incompatibility: more compatible than previously thought?. I will try every possibilities but it's better to design a system with clear knowledge
pDNA on Sun Mar 6 21:03:51 2011 said:
in principle it should be possible to maintaine both plasmids stable when selection pressure is used ...i must confess i do not fell really cosy with the idea using two plasmids with the same ori and therefore i avoid it when possible (and most of the time it is since we have several origins from which we can choose.
You can think about replacing the pET for a pETcoco™ ...see the corresponding info here.
Regards,
p
Why do you need two plasmids with different copy numbers?
Quasimondo on Sun Mar 6 16:06:25 2011 said:
I have a question regarding the compatibility of pBR322 ori in pET vector system and pUC ori in pUC19. As far as I know, they are basically the same except one mutation and the absence of rop gene in pUC19 so it has more copy number. In E. coli, if I want to maintain the 2 vectors in one cell with middle and high copy number, seems like only these ories can help. Actually I was suggested by one of my senior to do that and he also did the same thing. I'm going to construct a pET derivative vector with Amp resistance and a pUC - pACYC hybrid vector (with p15A ori replaced by pUC ori fragment) with Kan resistance. My senior used pET and a TOPO vector with both Amp and Km resistance and seems like they worked.
So, I wonder that the copy number of the pUC derivative vector would be reduced or not, since the rop gene presents on pET vector. The second thing is maybe this system will not stable, especially when I use BL21 as the host (rec+ strain), these ories can be exchanged.
Is there anyone here has experience with this kind of two plasmid system or can help me to make it clear? If you have any better suggestion for the system, you're very welcome, I'll very appreciate that.
You might want to know about the variety of Biobrick vectors described here:
http://partsregistry.org/Plasmid_backbones/Assembly
Also, the pDuet vectors are designed for simultaneous transformation, and are available in many different origins and resistances.