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

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Post doctoral positions in Structural Immunology
From: Steve Almo almo {- at -} MEDUSA {- dot -} BIOC {- dot -} AECOM {- dot -} YU {- dot -} EDU
Date: 2007-04-19

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Subject: Re: Protein expression in Minimal media (M9)
From: rgccri {- at -} CID {- dot -} CSIC {- dot -} ES rgccri {- at -} CID {- dot -} CSIC {- dot -} ES
Date: 2007-04-19

Subject: Re: Protein expression in Minimal media (M9)
From: jwallen {- at -} CSB {- dot -} WFU {- dot -} EDU jwallen {- at -} CSB {- dot -} WFU {- dot -} EDU
Date: 2007-04-19

Manish,

I also had a similar problem getting cells (C41 (DE3) in my case) to grow
in minimal media. To get around this problem, I took cells from an agar
plate and grew them in a small volume (5 mL) of the minimal media. Once
that culture got thick, I then inoculated 200 mL of minimal media with 0.5
mL of the 5 mL culture. I would let this grow, and I used this as my
overnight culture to inoculate my large flasks. Long story short, the
cells seemed happier adjusting to the minimal media in a smaller volume
first.

Cheers,

Jamie Wallen

> Hello everybody,
>
> Sorry for an offtopic question. I am trying to express a protein in M9
> minimal media for Selenomet incorporation. 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).
>
> It expressed poorly in BL21 (DE3) when grown in LB and thus decided to use
> RosettaBlue (DE3). It worked very well in LB, but having a hard time
> while expresing the same in minimal media using Rosetta Blue. Has anybody
> tried expression in minimal media using Rosetta Blue cell line? I am
> planning to try overnight induction.
>
> Any suggestions would be greatly appreciated.
>
> Thanks,
>
> Manish
>
>
>
>
>
>
>
>
> *************************************************
> Manish B. Shah, PhD.
> Postdoctoral Fellow
> Hauptman-Woodward Medical Research Institute
> 700 Ellicott Street
> Buffalo, NY 14203.
> *************************************************
>

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Post doctoral positions in Structural Immunology
From: Steve Almo almo {- at -} MEDUSA {- dot -} BIOC {- dot -} AECOM {- dot -} YU {- dot -} EDU
Date: 2007-04-19

Next message:
Subject: Re: Protein expression in Minimal media (M9)
From: rgccri {- at -} CID {- dot -} CSIC {- dot -} ES rgccri {- at -} CID {- dot -} CSIC {- dot -} ES
Date: 2007-04-19