Growth/expression in minimal media
hzhen at freeuk.com
Fri Apr 14 19:53:51 EST 2000
"Richard P. Grant" wrote:
> Thought I'd posted this once but it seems to have gone awol.
> OK, given BL21 DE3 [RIL] with a kanR plasmid, therefore selecting on
> Kan/Cam, I'm trying to grow labelled protein for NMR.
> Growth seems a little poor. Any tips for improving it?
> Currently in M9 (minimal media)+ 1 g/l NH4Cl, 0.4% glucose, 1 mM
> MgSO4, 0.1 mM CaCl2. I got hold of some Bacto yeast nitrogen base w/o
> aas but will this result in 14N-protein??
Very likely. Some of the components will go into amino
acids synthesis pathway and become incorporated into
protein. If you want to add that, you can only add a very
small amount. However, NH4Cl is not that expensive, you
might give it a go and how well/poor your labelling is. I
normally add some vitamins supplement (1000X
concentration/per litre: 0.5 g Choline chloride, 0.5 g Folic
acid, 0.5 g Pantothenic acid, 0.5 g Nicotinamide, 1.0 g
Myo-inositol, 0.5 g Pyridoxal HCl, 0.5 g Thiamine HCl, 0.05
g Riboflavin, 1.0 g Biotin), but that only have very minor
effect on growth. Your problem is likely to be the [RIL]
bit which affect the tRNA pool, and the kanamycin. One
possible thing you could do is to reduce the kanamycin. If
you want to splash out a bit, you can try the Celtone media
If you want to use M9, grow an overnight culture in M9, then
spin down cells and resuspend in labelled media to a
concentration of OD600 ~0.2. In this way you won't need to
wait too long before you induce the cells.
You can use 15N label for the overnight culture if you want.
> Richard P. Grant MAD Phil http://www.gerbil.org.uk/
> Please reply to rpg 'at' mrc-lmb.cam.ac.uk
> 'There's a fine line between genius and just farting around'
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