Cells with pET16b are not growing in liquid Medium

Dr. Peter Gegenheimer PGegen at UKans.edu
Fri Aug 14 19:09:18 EST 1998


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. 
                                   
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