Manish,

In practice, we have found that it is very helpful to grow up an
overnight starter culture in minimal media to acclimatize the cells for
growth under minimal conditions. (Cells transferred from LB to M9 do not
perform well for overexpression).  We inoculate 1 L of modified M9 (see
below) with 10 mL of an overnight culture (centrigued, washed, and
ressuspended in M9) in the same medium.

In addition--and even though it should not be required for many strains
of E. coli--we have found that he inclusion of thiamin, 5-10 ug/L,
greatly enhances the rate of cell growth, but it may still takeup to 8
hr for cells to grow to OD600 = 0.6 for induction. Overexpression
overnight (12-18 hr) may be required to get optimal cell density and
overexpression yield. Using this protocol we get excellent
overexpression yields of protein.

Cheers,

___________________________________________
Roger S. Rowlett
Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [log in to unmask]


-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
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Sent: Thursday, April 19, 2007 10:31 AM
To: [log in to unmask]
Subject: Re: [ccp4bb] Protein expression in Minimal media (M9)


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.
> *************************************************
>