Copper Dose Response of the Cup1 promoter using N4

Results

 

Title:

Quantitative determination of the response of the CUP1 promoter to varying concentrations 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 exhibits a linear relationship between [CuSO4] and the level of GFP expression when CuSO4 concentrations range from 0µM to 100µM. At concentrations of 100µM and higher the expression level of GFP will have reached a steady state and will remain unchanged despite increasing CuSO4 concentrations. [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

 

Tube n°	1	2	3	4	5	6	7	8
[CuSO4] (µM)	0	10	15	20	25	50	75	100

 

Each one of these tubes was inoculated with N4 from each starter culture in order to provide triplicates of each concentration 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 fluoremeter running the “RussGFP” protocol.

 

Result

For full data spreadsheet

èIGEM 240610 JH+SL PCup Induction.xslx

 

 

 

 

 

 

 

Figure 1 Plot showing the relationship between the average GFP expression and [CuSO4]

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Conclusion

The data suggests evidence of a dose dependent response. We can see from Figure 1that the relationship between the GFP levels and the copper concentrations is linear when the concentrations of CuSO4 range from 0µM to 75µM. At concentrations higher than 75µM the response seems to reach a plateau and the GFP levels no longer increase along with increasing copper concentrations. Figure 2 shows us that these characteristics appear for each individual culture.

The data supports the hypothesis that the response of the copper promoter is dose dependent for a defined range of copper concentrations. The data however indicates that this range is smaller than our initial hypothesis suggested and that a plateau is reached with concentrations of 75μM as opposed to the initial 100μM.

 

References

[1]. Gorman JA, Clark PE, Lee MC, Debouck C and Rosenberg M

Regulation of the yeast metallothionein gene

Gene, 48 (19860 13-22