expression/induction depends on container

hardies at BALIN.UTHSCSA.EDU hardies at BALIN.UTHSCSA.EDU
Mon Sep 12 09:43:19 EST 1994


H.T. Wright writes:

> 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.    

My first guess would be that the 500 ml culture grew faster because of better
aeration and was in stationary phase by the time you induced.  Lack of some
limiting nutrient would then limit yield.  What were you trying to induce:
a protein, RNA, DNA replication?  In general you want to induce in mid log
phase and turn your media into product, not cells.  Note that growing to
stationary phase, then refreshing the medium and inducing generally doesn't
work, because the cells down regulate and take a significant time to
recover.

My second guess would be that your sample from the 500 ml culture was bigger
or had more cells and that however you open your cells didn't work at the
higher scale.  Are you comparing your transgenic product to some endogenous
material to judge induction?

Good luck,
Steve Hardies, Dept. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu




More information about the Methods mailing list