Timed Induction of GFP Expression by the CUP1p-[GFP]

Aim

The aim of this experiment is to determine how quickly the CUP1 promoter is fully induced by set concentrations of copper by measuring the levels of GFP being expressed by the yeast cells.This is done using the CUP1p-[GFP] construct.

Hypothesis

The CUP1 promoter will rapidly become fully induced following exposure to set concentrations of copper. Once it is fully induced the level of GFP expression will stabilise and will no longer increase despite more time passing.

Protocol

A genomically integrated GFP gene under control of a CUP1 promoter in the construct CUP1p-[GFP], was used to characterise the control properties of this promoter. This construct was transformed into the yeast strain BY4741 for analysis.

Starter cultures of CUP1p-[GFP] in 5mL of SD medium + Raffinose were set up and incubated overnight. A 50mL flask containing SD Raff was then inoculated using the starter cultures and incubated overnight until the OD600 reached 0.3.

At t=0 a sample from the flask was put on ice to provide a background reading of yeast’s natural fluorescence without any inducer. Copper was then added to the flask at a concentration of 100μM. Samples were then taken every 20 minutes and put on ice. All samples were then normalised to an OD600 of 0.75 and were washed and re-suspended in PBS before being analysed in the fluorometer (the programme RussGFP” was used for the analysis).

Results

For full data spreadsheet
iGEM 2.6.10 JH+SL N4 Timed Induction.xslx

Conclusion

Figure 1 indicates that full induction was reached after approximately 80 minutes and that the level of GFP expression seems to reach a plateau after this and no longer increases. This supports our initial hypothesis.

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