By Levi Clancy for Student Reader on
Auxotrophs are unable to synthesize all their own biomolecules (nutrients, such as amino acids).
For example, a bio- mutant is unable to synthesize biotin. Auxotrophs can be studied to understand pathways for the synthesis of amino acids. Also, they can be used for other experiments (such as the Ames test and R-factor experiment).
Isolating a lysine auxotroph, from a mixture of auxotrophs and prototrophs, is a simple procedure. You can easily modify this procedure to isolate almost any specific kind of auxotroph.
Grow cells in media containing penicillin (which will kill dividing cells) and all amino acids except lysine.
Remove the penicillin-laced media and replace with media containing lysine
Screen for lysine auxotrophs
Further assays may be performed to determine the location of the mutation in the lysine synthesis pathway
Another, more efficient procedure is to:
Plate auxotroph/prototroph mixture on enriched GMA
Screen for tiny colonies
Pick tiniest colonies
Grow on GMA and YTA
Colonies able to grow only on YTA are auxotrophs
Auxotrophs form tiny colonies because there is only a small amount of complex media in enriched GMA.
This is an example of screening, because you distinguish between the two different colony types (auxotrophs and prototrophs) based on colony morphology.
However, if you want to isolate any cell exhibiting auxotrophy (from a batch of overwise prototrophic cells) then the procedure is different.
This can be helpful if you want to determine the pathway for synthesizing a certain compound. The procedure for outlining the biosynthetic pathway for lysine is given below. You will use E. coli as your start culture because WT E. coli are prototrophic.
Inoculate minimal broth with E. coli
Mutagenize culture, resulting in mixture of auxotrophs and prototrophs
Replace media with minimal broth + glucose + lysine
Plate onto enriched GMA
Pick tiny colonies, inoculate GMA and GMA+lysine
Colonies that grow only on GMA+lysine are lysine auxotrophs
Perform a test.