Posted 07 November 2011 - 11:25 AM
I would like to describe you briefly the situation with which I have come across during my last experiments.
I want to transform 2 plasmids in E.coli in order to co-express 2 different proteins. Each plasmid will carry different gene of interest of course, with 2 different promoters. Also the 2 plasmids have different antibiotic markers; one with ampicillin and the second with kanamycin. Just to simplify my description: 1 plasmid is pUC8 (lac promoter) and the other one pET29 (tet promoter-modified). My question is simply the following: can I maintain with their functionalities these 2 plasmids since they carry different selection markers? I am asking because the pUC8 plasmid has pMBI origin of replication while pET29 pBR322 origin of replication. In principle, pMBI origin arises from pBR322 and is considered to be of common replication mechanism. Therefore, perhaps these 2 origins of replications are incompatible for maintenance in the same E.coli cell. Or do you think that due to the fact that I already have 2 different selection markers, I will not encounter problems with co-expression.
Thank you very much for your time and your help in advance.
Posted 07 November 2011 - 01:00 PM
but ...Ampicillin is degraded rapidly and as soon as you lack selection pressure the plasmid with the amp plasmid will get lost (that's what i assume!)
to be honest ...i would do it differently
i would use the pUC8 and instead of the pET29 i would use a plasmid with a p15A origin of replication or a pSC101 (low copy is more than sufficient for expression using T7 promoter) ...the problem is that you'll have to generate such a plasmid yourself (replace the pBR322 ori of pET29 with a p15a) since i have no such commercial plasmid in mind at the moment (maybe some other forum member has).
Alternatively, you can go e.g. for the pETDuet plasmids (2 MCS for the co-expression 2 proteins form one plasmid) or the pACYCDuet (p15a ori and 2 MCS) ...both plasmids are compatible and in principle you can co-express 4 genes in total.
Hope this helps!
Posted 08 November 2011 - 07:20 AM
Posted 09 November 2011 - 01:02 AM