About 5-Fluoroorotic Acid (5-FOA)
Using 5-Fluoroorotic Acid (5-FOA) for the counter-selection of yeast is a common genetic screening method. Curing yeast strains of plasmids, plasmid shuffling, allelic replacement, and two-hybrid screens are methods that can employ the use of 5-FOA. Otherwise nontoxic to yeast, 5-FOA is converted to the toxic form (i.e., 5-flurouracil) in strains expressing the functional URA3 gene coding for orotine-5-monophosphate decarboxylase that is involved in the synthesis of uracil. Yeast strains that are phenotypically Ura+ become Ura- and 5-FOA(R) (resistant) after selection. The question of 5-FOA solubility is often raised by customers using ultra-pure (< 98%) 5-FOA powder because of its insolubility in water. Thus, we provide a 100X concentrated (100 mg/ml) 5-FOA solution in DMSO and a 2X concentrated (2 mg/ml) synthetic complete 5-FOA (i.e., 2X SC/5-FOA) in liquid medium containing yeast nitrogen base, ammonium sulfate, complete amino acids, and glucose. Both have been tested and validated on the basis of counter selection activity (see below).
|Format||Synthetic 5-FOA monohydrate powder or liquid.|
Counter selection of yeast using 5-FOA. Yeast strains that are auxotrophic for uracil (ura3-1) were tested for their ability to grow on 5-FOA containing media. Three strains were tested: wt alone (YZ1), wt with a URA3 marked low copy plasmid (YZ2), and a mutant strain with a deletion of an essential gene (?EG) that could not lose a complementing URA3 plasmid (YZ3).
From left to right, top to bottom are synthetic complete glucose medium (SC): 1. SC, synthetic complete no 5FOA; 2. Standard - SC-5-FOA (SC-5-FOA made from ultra-pure 5-FOA powder, 1 g/liter) 3. SC-5-FOA made from 100X 5-FOA solution; 4: SC-5-FOA made from 2X SC/5-FOA.
For each plate, Top: Yeast strain: YZ1 wild-type, Ura- (wt, ura-3-52), Right: Yeast strain: YZ2, wt carrying a low copy, URA3 plasmid alone, and Left: Yeast strain: YZ3: ?EG, containing the complementing plasmid (pRS316: EG, URA3, CEN). The counter selection against strain YZ3 was evident for all media containing 5-FOA with no 5-FOAR colonies evident (see left panels, YZ3: in plates 2, 3, and 4). Cells from control strains YZ1 and YZ2 were able to grow on 5-FOA media.