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I would like to find a method of increasing the biomass of my D2O cultures because my current method is not yielding enough protein. I would like to also minimize the amount of H2O in my culture.

My current methodology is a small scale growth of 3mL with 75% D2O and LB grown for approximately 9hrs. This culture is then used to seed a 20mL 100% D2O and M9 culture overnight and this entire culture is used to seed a 1L D2O and M9 culture.

Thanks for your time.

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It looks like you're very stringently avoiding $^1\mathrm{H}$. You may want to consider replica plating your transformations onto $\mathrm{D}_2\mathrm{O}$ plates (selecting for $\mathrm{D}_2\mathrm{O}$ tolerance earlier.)

I assume you're doing protein NMR and want "triple labeling." Depending on how specific your carbon labelling is, you may want to grow your starter cultures on triple labeled agal hydrolyzate (something like "Bioexpress.") You can buy some unlabeled stuff for fairly cheap (\$55 USD at CIL for 10 mL of 10X stock) and if your starters grow well you can spring for the same quantity of $^2\mathrm{H}$,$^{13}\mathrm{C}$,$^{15}\mathrm{N}$ for around \$405.

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  • $\begingroup$ A guy I talked to who was doing triple labeling said he had to give his bacteria several generations to adjust to increasing amounts of non-normal isotopes. I can't remember any other details, but you might not just be able dump the bacteria in a labeled media and make it work well. $\endgroup$
    – user137
    Commented Nov 30, 2014 at 6:22
  • $\begingroup$ @Ryan Thanks for the reply but it seems like this would not be a financially viable method for our lab. We recycle as much D2O as we possible can and to make agar plates with D2O would be wasteful for us. Additionally, we do use BioExpress and the labeling is not an issue. It just seems like our issue is with the amount of E. coli biomass. $\endgroup$
    – gravityassist
    Commented Nov 30, 2014 at 20:18
  • $\begingroup$ @user137 Do you by any chance know how many generations he grew his cultures? $\endgroup$
    – gravityassist
    Commented Nov 30, 2014 at 20:19
  • $\begingroup$ @gravityassist if you're already using Bioexpress that contradicts your stated use of M9. So I'm confused what you're trying. $\endgroup$
    – Ryan
    Commented Nov 30, 2014 at 23:40
  • $\begingroup$ @gravityassist I'm sorry, I don't. It was a 5 minute talk at a poster presentation over a year ago, so I don't remember much. $\endgroup$
    – user137
    Commented Dec 1, 2014 at 4:21

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