Quantitative determination of the response of the CUP1 promoter to varying concentration of CuSO4

Aim

The aim of this experiment is to characterise the CUP1 promoter present on N4 by determining whether it displays a dose response quality. The determined characteristics of this promoter can then be applied to the promoter present in pRS414 in order to allow more precise modelling of the switch.

Hypothesis

The CUP1 promoter ixhibits a linera relationship between the concentration of CuSO4 and the level of GFP expression when CuSO4 concentrations range from 0microM to 100microM. At concentrations of 100microM and hicher the expression level of GFP will have reached a steady state and will remain unchanged despite increasing CuSO4 concetrations. [1]

Protocol

A genomically integrated GFP gene under control of a CUP1 promoter was used to characterise the control properties of this promoter, this construct is referred to as N4 and was transformed into the yeast strain BY4741 for analysis.
Three separate starter cultures were prepared of N4 in 5mL SD medium. the following tubes were then prepared in triplicate.

Each one of these tubes was inoculated with N4 from each stater culture in order to provide triplicates of each concentratuion value. the cells were later harvested once the OD600 had reached 0.6. Triplicates from each culture were then loaded onto a 96 microtitre plate. The fluorescence of each sample was measured using a fluorometer running the "RussGFP" protocol.