Hi,
Is it possible to add 2 of the same genes under the same promoter to an integrative vector?
My goal is gene upregulation so I am adding a strong promoter to the gene and want to add 2 copies to upregulate even further.
For example promoter-gene-promoter-gene.
Is it possible without gene hybridization and competition for polymerase?
Thanks
The direct repeats tend to not be stable in E.coli during cloning. I would suggest you simply clone your gene into two different integrative vectors (would integrate in two different loci) then you can have two copies of the your gene. If you really need the same vector you can try two different strong promoters to reduce the homology and see if that works.
While I agree with the answer of Hanna Alalam for typical E. coli strains, most cloning strains are recA- strains which abolish homologous recombination. If you are careful to always use a recA- strain then you should be ok making the clone. Of course you will do need recombination if you intend to do an integration and while it is possible that recombination will be a problem, it should would work ok with care. There are lots of examples of duplicate genes in both E. coli and other orgaisms.
However I'm not sure you are necessarily going to see a doubling of your protein expression so you need to decide if it is worthwhile.
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