Lidstrom:media for methylotrophs

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What is the difference between MM1/MM2/MM3/HY?

  • They all have the same trace elements solution. The primary difference is the nitrogen source:
    • MM1/HY uses ammonium
    • MM2 uses nitrate
    • MM3 has no added nitrogen
      • MM3 is used when growing on methylamine, which releases N as it is metabolized
  • FYI, the trace elements are added after autoclaving for both liquid and agar formulations. Thus it is possible there could be contaminants before you start an experiment.

If all organisms assimilate N into biomass as NH4+ why do we give them MM3?

  • This is an interesting question, as the organisms must convert nitrate (NO3-) into ammonium to assimilate it. Two likely factors:
    • Ammonium is too toxic
      • In methanotrophs, it interferes with pMMO (methane assimilation enzyme). We don't have a tested hypothesis for why ammonium would be toxic to methylotroph.
    • They prefer to convert nitrate to ammonium (using NADH) to release energy, as they have an excess of NADH due to their metabolic strategy.

Why don't we have a media with nitrite?

  • It is toxic.

Why do we autoclave the MM1 mixtures separately before mixing them?

  • Precipitation occurs if you mix them before autoclaving. The precipitates cannot be resuspended by warming the media back up in the microwave and shaking it. -- Janet & Helen 2/2015.
    • update: The crystals did re-dissolve, and the media appeared fine.
    • MM1A made where both solutions were mixed prior to autoclaving worked great. - Janet & Helen 3/2015
    • See notes at Lidstrom:MM1_recipe

Media for matings: NB vs LB

  • Rich medias are generally toxic to methylorophs. When mating E. coli with methylotrophs you need to balance the need to feed E. coli and the fact that rich medias are toxic to the methylotroph.
    • When Mila mates diverse methylotrophs, she uses no rich media on the mating plate. Because E. coli is starving, a great excess of E. coli cells is used instead.
    • Sometimes a compromise can be struck by diluting the rich media in the mating plate.
  • AM1 is mated on NB, not LB. Since both are rich, why does this matter?
    • Mila suspects LB is richer than NB and hence more toxic to methylotrophs. It is a little hard to track down the exact recipes, but someone should to fact check this!
      • Nutrient broth/agar is a moderately rich, general purpose, solid medium that meets the nutritional requirements of many culturable bacteria. It contains beef extract, soy digest, and enzymatically digested gelatin to support the growth of a wide variety of chemoheterotrophic organisms. (source)
    • There are several common formulations of LB. Although they are different, they generally share a somewhat similar composition of ingredients used to promote growth, including the following: Peptides and casein peptones, Vitamins (including B vitamins), Trace elements (e.g. nitrogen, sulfur, magnesium), Minerals. (source)