T7 terminator seq needed

Dominic-Luc Webb molmed domweb at mbox.ki.se
Fri Aug 10 07:22:10 EST 2001


On Fri, 10 Aug 2001, Dr. Duncan Clark wrote:

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


We are trying with EGFP (humanized GFP), which Clontech claims
expresses well in E coli, although they did not specify if they
added any tRNA, as by introducing a second plasmid, to do so.
Several people have told me the codon usage should not explain
the low expression I have gotten.

What I have tried is RT 6 hours, 37C 3 hours at IPTG concentrations
of 0.1, 0.5 and 2.0 mM. Everyone I have asked felt that one of
these conditions should have worked pretty well.

Could I please ask which vector(s) you have used? Our's has same
promoter and Shine Dalgarno in same location as at least one
commercial prokaryote expression plasmid.


> 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.


Hmmm, I didn't know that they sold an IPTG inducible plasmid.


Cheers,



Dominic

Lab:
Department of Molecular Medicine
Endocrinology and Diabetes Unit
Rolf Luft Center for Diabetes Research
Karolinska Hospital L3
S-17176 Stockholm
Sweden
Tel: Int+46-8-517-74829
Fax: Int+46-8-517-79450
Internet Email: dominic at enk.ks.se
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