Cells with pET16b are not growing in liquid Medium

Keith Rand Keith.Rand at molsci.csiro.au
Sun Aug 16 23:18:16 EST 1998


In article <7opiGDf98QgB-pn2-x69EtZuYH4Wp at rnaworld.bio.ukans.edu>,
PGegen at UKans.edu wrote:

> On Wed, 12 Aug 1998 15:29:59, svetlov at oncology.wisc.edu (Vladimir 
> Svetlov) wrote:
> 
> > In article <35d18f5b.25553585 at news.ruhr-uni-bochum.de>,
> > Matthias.drosten at rz.ruhr-uni-bochum.de (Matthias Drosten) wrote:
> > 
> >                  
> >  > Hello,
> >  > I have got the following problem:
> >  > 
> >  > After transforming a ligation with pET16b and an insert into XL1 blue
> >  > I get about 60 colonies (7 colonies at the religation control) on TBY
> >  > + Amp.   The grew very slowly.  Transferring them into liquid medium
> >  > they are not growing at all (at the moment nothing had happened for  2
> >  > Days.). Transferring them to an agarplate they are growing again.
> --- From where are you transferring them (not from the liquid 
> culture?)?
> >  > 
> >  > Has anyone an idea to solve this Problem?? I want to isolate the
> >  > plasmide to verify the ligation.
> > 
> > 
> > Such things happen if the insert's message is toxic for the cell. pET
> > vectors have substantial leakage of transcription even without induction in
> > XL1, DH5a and JM109 cells. If you can get some growth on the plate, you can
> ..snip...
> 
> pET 16 does not have a lac promoter, so expression of a cloned gene 
> is not inducible in any host cell which lacks a cloned gene for T7 
> RNA polymerase. 
> 
> I just looked at the Novagen catalogue; pET16 vectors do have a lac 
> operator following the T7 promoter. This means that transforming 
> pET16 into any cell with a chromosomal lac gene (even an 
> alpha-complementation strain like XL1 Blue) will induce the lac 
> operon (without IPTG present). This could result in slower growth of
> the cells, although we've never noticed this. 

If this is the case, maybe 1% glucose would help (?)

-- 
Keith Rand,  Sydney Australia



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