expression/induction depends of container

Marc A. Goldstein magoldstein at ucdavis.edu
Sun Sep 11 23:10:12 EST 1994


In article <01HGWXSEL5JMA74DNS at Gems.VCU.EDU>, XRDPROC at Gems.VCU.EDU wrote:

> Can anyone who knows something about bacteriology explain the 
> following?  We inoculate 510 ml of our medium in a 2.8l Fernbach flask
at about 
> 100 or 200:1 from an overnight culture of our heat-inducible plasmid-carrying 
> E. coli strain in the same medium.  We immediately remove 10 ml into a
culture 
> tube with an aeration plastic cap, and the remaining 500ml of inoculated 
> culture remains in the Fernbach flask, covered either with aluminum 
> foil or with a cotton/cheesecloth stopper.  Both cultures are grown to high 
> density with vigorous shaking at 30 degrees and simultaneously induced by 
> raising the temperature to 42 degrees, which is maintained for 4 hours, 
> sometimes longer.  When run on gel, samples from these cultures show that the 
> 10 ml culture was strongly induced, while little or no induction occurs for 
> the larger culture in the larger flask.    
> 
   This is a common effect.  The growth conditions that the different
portions are subjected to are not identical.  Probably, the amount of
dissolved oxygen/carbon dioxide is at the root of this.  It could be that
the 10 ml culture, in a tube, was growing relatively slowly. Induction in
slowly growing cells often leads to _greater_ protein accumulation, due to
a lessening of the toxicity effects of certain heterologous proteins. 
Most protocols suggest growing at lower temperature (or other
growth-slowing conditions) in the event of poor protein expression. This
example may not be possible for you, as your induction is temperature
dependent.

   So try some easy fixes: Reduce the amount of oxygen getting into your
fernbach flasks by covering the top partially or fully with parafilm. 
Slow the shaking speed. Also speed it up.  Fernbachs have no baffles on
the bottom.  Try growing up your cells in baffled flasks (bellco, kimax). 
Go from high oxygen to no oxygen, to see if that makes a difference. 
Also, try harvesting at different times, from 1 to 6 hours post-induction.

   Best of luck.  Post your result, to let others know what if anything,
solved the problem.
________________________
Marc A. Goldstein
Section of Plant Biology
UC Davis
magoldstein at ucdavis.edu



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