Complementation experiment of yeast single knockout strains with mtnBD fusion gene from Tetrahymena.
Cells were grown to late exponential phase in −Leu +Met liquid media, transferred to −Leu−Met for an overnight to deplete internal Methionine pool, and serial dilutions for each strain were prepared in a 96-well plate with 1×108 cells/ml, 1×107 cells/ml, 1×106 cells/ml, 1×105, 1×104 cells/ml, and 1×103 cells/ml. 3 µl of each diluted culture was spotted with a 96-well pin replicator onto (A) −Met−MTA (negative control plate). (B) +Met plates, (positive control). (C) −Met +MTA (5 mM) (experimental plate). The strains assayed are: 1. mtnBΔ + pGREG505/SYN-MTNBD; 2. mtnBΔ + pGREG505; 3. mtnCΔ + pGREG505/SYN-MTNBD; 4. mtnCΔ + pGREG505; 5. mtnDΔ + pGREG505/SYN-MTNBD; 6. mtnDΔ + pGREG505; columns 7–12 are replicates of columns 1 through 6. After four days, none of the six yeast strains grew on the negative control plate. All six strains grew on the positive control plate. Only the strains transformed with pGREG/SYN-MTNBD (columns 1, 3, 5, 7, 9, 11) grew in the experimental plate.
