Chemically defined media required

Aawara Chowdhury via methods%40net.bio.net (by aawara from pontiff-playground.org)
Mon Apr 13 09:13:28 EST 2009


In <fHGEl.42978$_R4.11826 from newsfe11.iad>,
 DK <dk from no.email.thankstospam.net> wrote:

>>we would like to switch over to chemically defined media from complex media
>>so as to increase yield. can anyone suggest how can we go about it as the
>>media constituent we found on the net gives trace metals content to be adde=
>>d
>>but not of vitamins. kindly help.

First off, it is highly unlikely that a chemically defined medium, such as
M9 minimal media, suggested by DK will increase yield, which appears to
be your primary concern - as opposed to increasing yield - unless by the
latter you mean soluble protein.  For solubility, I agree with DK's
suggestion that you should switch to a chemical inducer, rather than 
temperature induction - what are you using - a ts mutant of lambda cI?

That system, now several years old, typically placed two copies of 
lambda OR1 immediately after the tac promoter or the lacUV5 promoter.
At 30oC, cI was bound to the operators blocking the elongation of
transcripts that initiated at tac or lacUV5; at 42oC cI was inactivated,
permitting transcript elongation.

While the lacUV5 and tac promoters are strong promoters for E. coli RNAP,
they are weak compared to the activity of T7 RNAP on the T7 promoter.

So re-cloning your protein into one of Bill Studier's pET vectors, and
inducing them in a strain that inducibly expresses T7 RNAP, such as
BL21(DE3) - or one of its many derivatives, will significantly increase
your yield.  In addition, as Dima points out, you can induce at much lower 
temperatures, which in turn will likely increase solubility.

> Google "M9 minimal medium". If the protein is insoluble, heat induction 
> is likely not a way to go. You'd be better off switching to IPTG induction
> and expression at 16C. Vitamins are not required for a typical expression
> strain. 

Yes - this is correct.  One other suggestion.  What about just denaturing
and renaturing the protein that is in inclusion bodies?

AC
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