T7 Expression System

[TJCooper]

Good evening,

 

I am currently a first year molecular biology student at university. I just have a question and I hope that it's okay to ask here. I currently have a simple expression system setup within E.Coli K12 strains, using a phagemid with Tet-R/Amp-R genes, and my gene of interest inserted downstream of the T7 promoter however there is no T7 terminator present as far as I know. I plan to introduce a heat-inducible plasmid containing the gene for the T7 RNA polymerase under the control of an E.Coli promoter into the cells, and induce it in the presence of labelled amino acids, and Rifampicin to inhibit endogenous RNA polymerases in the hope of translating primarily the gene of interest, labelled, and easily purified. My question is - knowing that the T7 RNA polymerase can be highly processive, is it likely to also transcribe the Amp-R/Tet-R genes and thus make it somewhat harder to purify the gene of interest?

I plan to assay a mutant protein against it's wild-type form and compare the activities thus I require a high purity as I need to know the concentrations of each to be able to compare the result from the bioluminescence assay.

Also do phagemids simply contain bacterial promoters too so that Amp-R/Tet-R can be expressed?

Is there any other way I can express and purify this protein (it's an adenylate kinase) in vivo, rather then simply using the T7 in-vitro system. Thanks,

TJ Cooper

Edited December 31, 2011 by TJCooper