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Re: [ccp4bb] Protein expression in Minimal media (M9)
 

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CCP4bb <-- 2007 <-- April 2007 <-- 19 April 2007
Previous message:
Subject: Summary - Stop Refmac from refining B factors?
From: Eva Kirchner eva {- dot -} kirchner {- at -} UNI-TUEBINGEN {- dot -} DE
Date: 2007-04-19 		Next message:
Subject: Re: Protein expression in Minimal media (M9)
From: Eric Toth etoth001 {- at -} UMARYLAND {- dot -} EDU
Date: 2007-04-19


Subject: Re: Protein expression in Minimal media (M9)
From: "John A {- dot -} Newitt" newittja {- at -} COMPUSERVE {- dot -} COM
Date: 2007-04-19
At 10:34 PM -0400 4/18/07, mshah@HWI.BUFFALO.EDU wrote:

>
>Hello everybody,
>
>Sorry for an offtopic question. I am trying to express a protein in M9
>minimal media for Selenomet incorporation.

What is your carbon source, glucose? glycerol?. Are you sure that you
have the right concentration of the carbon source in the medium?


>When grown in LB this protein
>expressed very well and got good crystals. Diffraction was upto 2 A. I am
>having a hard time expressing the same protein in Minimal media. It took
>nearly 24 hours for the OD600 to reach ~ 0.4 before inducing with IPTG in
>minimal media and eventually got no protein expression. It looks like the
>cells are not growing or taking very long to grow. The cell line I am
>using is RossettaBlue (DE3) and the plasmid is pET19b based (Novagen).

I haven't used that strain so I don't know if this matters, but some
strains have mutations that require vitamin supplementation in the
medium to improve the growth rate. Check the strain genotype.
Supplementing with amino acids (minus Met) should also improve the
growth rate.

- John

CCP4bb navigation

CCP4bb <-- 2007 <-- April 2007 <-- 19 April 2007
Previous message:
Subject: Summary - Stop Refmac from refining B factors?
From: Eva Kirchner eva {- dot -} kirchner {- at -} UNI-TUEBINGEN {- dot -} DE
Date: 2007-04-19 		Next message:
Subject: Re: Protein expression in Minimal media (M9)
From: Eric Toth etoth001 {- at -} UMARYLAND {- dot -} EDU
Date: 2007-04-19


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