wrong orientation of ligated fragment

Sean Yoder syoder at chuma.usf.edu
Sun Jun 6 09:37:33 EST 1999


     Is the gene from a prokaryote?  If so if the promoter is intact it may be
expressed in E. coli fairly efficiently.  I doubt a eukaryotic promoter would
allow transcription though...but I'm just learning this stuff so take ir for what
it's worth.

Sean Yoder

Microbiology Graduate Student
University of South Florida

Yuji Tai wrote:

> I am wondering if a mammalian expression vector in which a toxic
> fragment inserted still affect the bacteria viablity. Does the CMV
> promoter work in bacteria?
>
> > I presume that you have confirmed this by sequencing.
> > Anyway, there is nothing much one can do under the circumstances (single
> > site ligation, I mean). What is concluded normally is that the esxpressed
> > polypeptide in the correct (required) orientation is cytotoxic....
> > Suggestion:
> > clone the fragment in a bare-bone vector that does not have any leaky
> > promoter and has a BglII site along with others. Cut it out of this clone
> > with BglII and another RE (X). Clone it directionally into your expression
> > vector at BglII/X sites.
> > However, if the expressed prtoduct is indeed toxic to the cells, you are no
> > further along....
>
> Yuji Tai
> ytai at kms.ac.jp
> Kagawa Medical Univeristy
> http://www.kms.ac.jp/~physiol1/staff/ytai




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