I'm wanting to express 2 proteins (A,B) into a vector and transform cells with this.
In the distributed construct strategy, each insert is cloned into a different, compatible vectors that can be transformed into a cell. Rather than 2 DNA inserts (AB) in the same vector, and then cloned together in a parallel step, What is the advantage of such a strategy? Won't there be an uneven amount of A and B that has been uptaken by the cell as they are located in different plasmids/vectors?
Refer to the diagram below.
First of all, the strategy in B is only for bacteria and not eukaryotes unless you introduce specific signals like an IRIS signal. Secondly, B only establishes similar transcription for the two genes, but not necessarily similar translation. So it is unlikely that either will guarantee precisely the same amount of both proteins, although admittedly B is likely to be more similar. The main advantage of C is that it may be easier to make the construct, and easier to for example make a bunch of mutations in one and not the other.
Often you don't need the same amount of both proteins unless it is a heteromultimeric protein. But both approaches do work and the choice really depends upon the precise goals and details of the projects.
Method A can have issues with restriction site compatibilities between the MCS of the plasmid and the first insert (especially with longer sequences). Method B needs a 2A peptide cleavage signal if being translated as a single peptide or internal ribosomal entry sequence. Method B can have low yields of transformants if using conventional restriction digest with ligation (as opposed to recombineering or SIRA). Method C has the potential problem of not all plasmids being transfected into the same cell so can have low yields of double transformants. If your plasmids have a functioning origin of replication in the host then the copy numbers should stabilize after a few passages.
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