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6.3: Auxotrophs and selective media

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    17526
  • The met mutants are Met auxotrophs, meaning that they are unable to grow in media that does not contain Met. Auxotrophs are microorganisms that are unable to synthesize an essential nutrient because of a gene mutation. Many laboratory strains carry multiple mutations that interfere with the synthesis of essential nutrients. For example, because the BY4742 strain carries mutations in the HIS3, LEU2, LYS2 and URA3 genes, the strain will only grow in media containing histidine, leucine, lysine and uracil. Auxotrophic strains have many uses in genetics. Researchers often use auxotrophic strains as hosts for plasmid transformation (Chapter 12). The plasmids used for transformation carry functional alleles of a gene that is defective in the host strain, making it possible to select transformants by their ability to grow on media lacking the essential nutrient.

    Synthetic media are an essential tool for culturing and studying auxotrophs, because all of the components are defined. Yeast researchers have developed a variety of different formulations for synthetic media. All synthetic media contain a carbon source (usually D-glucose), a nitrogen source, and essential vitamins and minerals. The vitamins and minerals are usually purchased
    in a formulation known as yeast nitrogen base (YNB). The supplements added to synthetic media can be tailored to support or select against the growth of particular genotypes. In this course, we will use Yeast Complete (YC) medium that supports the growth of most S. cerevisiaestrains. The growth rate of wild type strains in YC is somewhat slower than that in rich media like YPD, but the strains are viable for long periods of time. The table on the following page shows the composition of YC, which includes a rich supply of amino acids and nucleotide bases. In addition to the complete YC medium, we will also use selective media in which some of components have been left out. For example, in this lab, we will use YC-Met “drop-out” media, which contains all of the YC components in the following table, except methionine.

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    *YNB is a complex mixture of vitamins, minerals and salts. Final concentrations in YC: Vitamins (μg/liter): biotin (2), calcium pantothenate (400), folic acid (2), inositol (2000), niacin (400), p-aminobenzoic acid (200), pyridoxine hydrochloride (400), riboflavin (200), thiamine hydrochloride (400).
    Minerals (μg/liter): boric acid (500), copper sulfate (40), potassium iodide (100), ferric chloride (200), manganese sulfate (400), sodium molybdate (200), zinc sulfate (400).
    Salts (mg/liter): potassium phosphate monobasic (1000), magnesium sulfate (500), sodium chlo- ride (100), calcium chloride (100).
    (Source: http://labs.fhcrc.org/gottschling/Ye...tocols/yc.html)