Hi,
I am looking for an expression plasmid to be used in combination with pET15b or pET26b. I need to express two potential protein partners in E coli. I prefer to do the tests with two vectors, because I plan to run a combinatorial approach, since each protein means in fact 10-20 different sequences. Therefore, it should be faster/easier to carry out simultaneous transformations with couples of plasmids than preparing by molecular biology all necessary vectors containing the many combinations (100 to 400) of the 2 ORFs.
Since I have already my ORFs in either pET15b or pET26b-based vectors, the best would be to transfer them to a vector with a different replication origin and resistance marker (not Kan or Amp) -- by the way, I would prefer to use the same induction approach than pET vectors, based on pET T7/Lactose promoter -- This could be done by transferring just the ORFs using couples of restriction enzymes. There are many possibiities, depending on whether promoter/terminators are transferred aswell or not. That is why I schematically show how they are organized in each one of my plasmids:
1) --------/BglII/--T7promoter---LacOperator---/XbaI/--/SwaI/--RBS---/NdeI/---ORF--/NotI/--tag1--translationSTOP--/BamHI/---/XhoI/---/BlpI/--T7terminator------- (KAN resistance)
2) -------/BglII/--T7promoter---LacOperator--/XbaI/--/MfeI/---RGS---/NdeI/---ORF--/NotI---/tag2--translationSTOP---/SalI/---/AvrII/--/XhoI/---/BamHI/---/BlpI/---T7terminator-------/HindIII/------- (Amp resistance)
If you know of any available vector that might be appropiate for my purposes, I would be extremely happy to know.
Thank you very much in advance for your help,
With kind regards,
Luis
Luis, I just saw this question. Just use a pET vector with a different drug marker. The strong selection is enough to maintain two plasmids with the same control system. We do this al the time with pET15 and 28 for example.
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