Hello Everyone,
Does anybody use a Trp based expression vector?
I had a few questions.
1. Are there any such vectors commercially available? If yes, please suggest the companies selling them.
2. I've already found sequences and papers regarding the trp based expression vectors. All of them mostly talk about inserting the promoter/operator region and the SD site in a pBR322 type backbone. But none talk about the transcriptional termination site. Is a transcription termination sequence important to be cloned at the downstream in the vector?
Thanks in advance.
Pankti
You are most likely reading very old literature. The Trp promoter is a fairly strong promoter and can be used, but it is difficult to regulate because you would need to grow in the presence of Tryptophan to grow in the repressed state (non-expression) and then remove the tryptophan when you want to induce the cells for expression. A hybrid promoter (TAC) was developed to make this simpler to do by inducing with IPTG. However there are currently much better and easier to regulate promoters for expression in E. coli and those are nearly universally used. I doubt you can find any commercial vectors any longer that use the Trp promoter and I can't think of any reason you would wish to use them.
In most situations you don't need to worry about transcription termination.
Don't use a trp promoter. As Michael points out, there are better alternatives out there. Although you can induce it even in the presence of tryptophan by using beta indoleacrylic acid, the real problem with pTrp is that the number of tryptophan repressor molecules per cell is not high enough to guarantee repression in medium copy number vectors (such as pBR322-derived plasmids). You can overexpress the repressor, but then you run into other problems, as it has operators in other aminoacid biosynthesis operons.
Also, you do need to include a transcription terminator. Run-off transcription interferes with antisense RNA-based replicons and may lead to plasmid instability, which is a big deal in some settings (though not in the lab).
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