[Microbiology] High cell density E.coli auxotroph fermentation

Duncan Clark via microbio%40net.bio.net (by blackhole from abuse.plus.com)
Fri Jan 28 05:20:43 EST 2011


Hi Folks,

I need to grow a Met- Trp- E.coli in a defined media with addition of 
Met and Trp. I would much prefer to have used BL21 or a prototrophic K12 
but the expression is just that much better in that particular auxotroph 
strain.

What I am struggling on finding is how much amino acid does one actually 
need to add per litre if I say we will get up to say  5x10E10cells per 
ml.

According to NEB's protein data info at the back of their RE catalogue, 
total protein at a cell density of 10E9 is 0.15g so at my cell density 
that will be 7.5g with a theoretical wet weight of roughly 45g/litre. We 
may even double or triple that in a fed batch fermentation.  If we say 
roughly 5% of each amino acid then we would have needed a minimum of 
0.375g each of Met and Trp per litre at the 5 x 10E10cells/ml.

Is that a sensible way to look at it and does that aa figure make sense?

Thanks for any insights.

Duncan

-- 
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.


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