Chris Larsen (clarsen at bimcore.emory.edu) wrote:
: We're trying to express a protein in minimal media, and I have no idea how long this usually takes. The problem is to keep all sources of nitrogen (but 15N ammonium chloride) out of the mix. Should this expression be takin 36 hours to reach OD600nm=0.5 ?
36hrs is a long time - I usually reckon on 5-8hrs from a 1% innoculum
(rich medium) to an OD of ~0.5, then grow O/N to a final OD of maybe 2.5
tops. Its worth playing around with different cell lines (I use TG2 and
JM109 - for different plasmids, one or other works better), with different C
sources (glucose & glycerol for example), and maybe with different N sources
(N sulfate ?). Keeping all 14N sources out isn't too bad - even the N in the
vitamin mix makes no odds. A 1% innocullum in rich medium is ok too.
: Has anyone tried to do this? I'm curious to see if the levels of expression are similar to that obtained in LB (rich) media.
My expression dropped from maybe 90mg/L in 2xTY to ~30mg/L in M9 - I get
the feeling this is fairly typical.
: Thanks,
: Chris Larsen
Ben
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Ben Davis,
MRC Protein Function and Design,
Cambridge, UK
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"They can make me do it, but they can't make me do it with dignity."
