Hi all,
I am designing a dual expression on the same vector (pET28a vector). I have successfully cloned the first gene at the multi-cloning site of pET28a in the first step. To cloning the second gene, I am going to replace the flanking region between restriction site of Bgl I and Tht111 I with a new sequence that designed for expression of the second gene (using T7 promoter). However , analysing this flanking region, I found it contains some elements included partial Rop and Tet. I don't know how important of this region to the activity of the vector, can we remove it off the vector?
Thank you for you suggestion.
Do you just want to co-express the two genes? If so, you may want to use the polycistronic vectors from Dr. Song Tan's lab [see Protein Expression and Purification 21, 224-234 (2001)]. I am not quite sure, but the region between BglI and Tth111 doesn't seem to contain important sequences for plasmid propagation.
I agree with Hua Xiao: if possible, try to get your hands on a bicistronic expression vector like pETDuet. If you want to tinker with it, you may engineer it such that it encodes a Tev protease cleavage site to remove the His-tag.
Thanks @ Hua Xiao and Pierre Béguin,
I aim to produce the specific recombinant protein by gene 1 and the RNA molecule by gene 2 in the same cell. Therefore, polycistronic vectors will not work in my experiment.
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