*4.7. Tolerance Assay and Growth Curve*

The final pRS-416-GFP vectors cultured in URA medium were diluted until the OD600 value was 0.1, and were incubated again until the OD600 reached 0.3. The cell culture was then five-fold diluted and treated with 75 μM Cd, 75 mM Hg, 100 mM Al, 50 mM Cu, 100 mM Co, 1 M NaCl and 2 M mannitol, respectively, and was incubated at 28 ◦C for five days [45]. No treatment was added for the control. The cold and heat stresses were applied at 4 ◦C and 38 ◦C, respectively, for 2 days before being transferred to a place at 28 ◦C to for 3 more days. The photos were taken after five days of incubation, and the experiment was repeated three times. The S1fa overexpressing yeast cells without and with the 1 M NaCl treatment were grown at 28 ◦C in liquid URA culture medium, and diluted until the OD600 value was about 0.1. The cells were incubated again, and when OD600 value reached 0.3, the OD600 was recorded every 2 h to prepare the growth curve of the cells [45].
