Hello,
I am currently running some experiments that require me to grow up Salmonella Typhi to express the Vi capsule. Currently due to safety restrictions I only have access to using the vaccine strain M01ZH09 which is deficient for synthesis of aromatic amino acids.
Does anyone have any experience with supplementing growth medium with amino acids? As in what concentrations should I use, also if you have any recommended suppliers? I am hoping this will improve Vi expression so I can use the bacteria for my assay.
Thank you very much for any help in advance!
Mari
LB is a rich medium have you tried to grow them in it without supplement, you can always make 2x LB to enrich it everything should grow in it.
Hi Mari,
I agree with Jaroslaw, LB should be able to support the growth of amino acid auxotrophs. However, using 2x strength might cause osmotic stress as it double the amount of salt in the medium (again depends if using Miller or Lennox LB).
If LB doesn't work well, I would recommend adding around 1mM of each aromatic amino acid. Our lab makes up a synthetic cystic fibrosis medium (SCFM) all the time and it supports rich growth of enterobacteria and respiratory pathogens. It is 0.1 - 1 mM for the aromatic amino acids.
Hope this helps!
Andrew .... what strain of P.a are you working with ?
I have a problem with "green" phenotype of Pa culture ... sometimes it green sometimes not ... any tricks to have them always green?
Jaroslaw Krol Our lab uses P. aeruginosa PAK, PA14, and PAO1. But I don't work with them, I work on Burkholderia. Others in the lab always culture it at 37oC in LB, brain-heart infusion medium, or King's B medium. Apparently, King's B medium is the best for getting the pigment production.
hmm... We have tried that too, not really working well.... at least my student said that :) Thanks
Hi!
Mari Johnson , have you tried supplementing the broth with Casamino acids? (Provides almost all essential amino acids but tryptophan)
I work with Burkholderia, I have used it to increase the amount of biofilm obtained and it has worked very well.
Hope is not too late!
Hi @ Mari Johnson,
I also suggest to try with Casamino acids, as advised by Laura Beltina León. Please try.
Hi Mari,
can you be more precise on which amino acid auxotrophies your strain has? Can you opost the complete genotype?
It is correct that Casamino Acids lack Trp. So if your strain needs Trp this is of not much use. If you need I can supply you with a list of ingredients from a typical analysis of its contents. As concerns concentrations: Why dont you simply test how much is needed for optimal growth. It is a one day experiment and you will know.
Best Jürgen
Hi everyone, thanks we found adding an aro mix to the broth worked, using casamino acid! :)
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