T7 terminator seq needed

Dr. Duncan Clark news at hgmp.mrc.ac.uk
Fri Aug 10 04:19:14 EST 2001


In article <Pine.GSO.4.33.0108091711380.24654-100000 at mbox.ki.se>, the
eminent Dominic-Luc Webb molmed at Stockholm University wrote
>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?

I've used a T7 expression vector for many years and it does not have a
transcription terminator. I can quite happily get expression up to at
least 50mg/litre of certain prokaryotic proteins. A terminator may help
increase this but it isn't a necessity.

I would tend to look at codon usage, induction conditions and the
promoter-rbs area for possible problems. 

Maybe a year or two ago I was given a Clontech GFP plasmid that runs off
a lac promoter and it fluoresces brightly when grown with IPTG. Under a
T7 promoter I would expect there to be even better expression so I find
it a little odd that you see so little. Having said all that I have no
other experience with GFP but a lot with T7 expression.

Duncan
-- 
Homogeneous Fluorescent Reporting Systems for Real-Time Quantitative PCR: 

Optimisation, Probe Technology & Future Systems

4-5 September 2001
King Alfred's College, Winchester, UK 

http://www.dstl.gov.uk/html/specialisations/index.htm




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