'Leaky' IPTG inducible promoters...

Simon Twigger mbxsnt at unicorn.nott.ac.uk
Wed Aug 3 07:58:17 EST 1994


Hi there,   (PS  My  space  bar is playing up,  hence all these double
spaces!)

I have a gene cloned into plasmid pKK233-2 which I assume is an earlier
version  of pKK233-3 that Pharmacia sell at present. Gene expression is
inducible using IPTG and I get about 1mg of product/litre of bacteria
following induction.

I did an analysis of bacterial extract before and after induction and found
that the actual induction seemed to make little difference. Using
antibodies to detect the product, there was a large amount of product prior
to induction and not a great deal more after induction.

My induction is as follows:  Grow up 10ml overnight, add  to 990ml of fresh
media, grow  for 2hrs (mid-log) add IPTG to 0.1mM, leave to   induce for
4hrs then lyse by sonication.  If i sample the 10ml overnight and also
after the induction step there is little difference  in  product levels. 
The total  protein  levels in each  sample appear the same  on coomassie
gels.

The promoter used in pKK233-2 is Ptrc. I know other promoters, especially
the T7 promoters, are known to be leaky and you get certain amounts of
background expression without IPTG.

Has anyone had similar problems with pKK vectors?

Does anyone know of any literature discussing these leaky promoters so I
can mention it in my thesis to prove its not just me!

Any help would be most appreciated.  

      Thanks in advance,


							Simon.


==============================================================================
Simon Twigger                                  =  
University of Nottingham Biochemistry Dept.    =  
E-MAIL     mbxsnt at unicorn.nott.ac.uk           = 
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