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

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CCP4bb <-- 2007 <-- April 2007 <-- 19 April 2007

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Subject: Re: I/sigma in XDS output
From: Kay Diederichs kay {- dot -} diederichs {- at -} UNI-KONSTANZ {- dot -} DE
Date: 2007-04-19

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Subject: Re: Protein expression in Minimal media (M9)
From: Roger Rowlett RRowlett {- at -} MAIL {- dot -} COLGATE {- dot -} EDU
Date: 2007-04-19

Subject: Re: Protein expression in Minimal media (M9)
From: artem {- at -} XTALS {- dot -} ORG artem {- at -} XTALS {- dot -} ORG
Date: 2007-04-19

Manish,

Assuming that you need the minimal medium for Se, have you tried the
regular heavy atom derivatives? You already have diffracting crystals...

M9 medium can be slightly difficult. However, no one says that you *have*
to use minimal medium such as M9 (again, assuming that you're doing
Se-Met). There are numbers of defined media compositions out there, many
with very good growth characteristics.

Finally, you can spike the M9 with a small quantity of yeast extract. This
will give your culture a decent initial boost and the amount of normal
methionine in the extract should be relatively small so it will all get
eaten up while the cells are still in early log phase.

Regards,

Artem

> 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 <-- 2007 <-- April 2007 <-- 19 April 2007

Previous message:
Subject: Re: I/sigma in XDS output
From: Kay Diederichs kay {- dot -} diederichs {- at -} UNI-KONSTANZ {- dot -} DE
Date: 2007-04-19

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Subject: Re: Protein expression in Minimal media (M9)
From: Roger Rowlett RRowlett {- at -} MAIL {- dot -} COLGATE {- dot -} EDU
Date: 2007-04-19