T7 terminator seq needed

Dominic-Luc Webb molmed domweb at mbox.ki.se
Thu Aug 9 10:33:46 EST 2001


I deeply appreciate all the help from the several members
of the list regarding my attempts to express protein in
E coli. I recently got the first little glimmer of hope
when I detected a small EGFP fluorescence on the
flourescence spectrophotometer expressed from our plasmid
bearing the T7 promoter and now having a Shine Dalgarno
(SD, AGGAGG) sequence.

I need to move on to perfection... Looking at the
difference between our plasmid and the commercial
ones, like pET, it seems that we are lacking a
T7 transcription terminator (T7 Tf) sequence. Adding
this may not be as trivial as just using the QuickChange
kit as we did for getting the SD sequence, it seems. The
T7 Tf sequence described on the Novagen plasmid map shows
over 40 bases!!! Wow...

Now I have some questions...

Do I really need so many bases for a fully functional
T7 Tf?

Could someone direct me to a "known" functional
sequence that will serve this function and where
it should be optimally located relative our gene
we are cloning?

Given that our present plasmid lacks this (it started
its life as a mammalian system using the T7 promoter
only for sequencing), is it common knowledge that the
lack of the terminator sequence can result in drastic
reduction in mRNA transcript, thus explaining my low
protein expression?


Cheers,


Dominic




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