Confirmation Using Microscope and Fluorometer Analysis that pRS414 was Not Expressing CFP

Aim

The aim of this experiment is to determine whether the pRS414 construct can express CFP in the presence of CuSO4. The detection of CFP using the fluorometer and the microscope would confirm whether the construct as a whole was functional or not.

Hypothesis

The observed lack of CFP expression is due to experimental errors and using appropriate medium and proper inducing agent, the pRS414 construct will express CFP.

Protocol

The Cyan filters on the fluorometer were first checked to ensure that they could detect CFP. Pacific Blue beads (a fluorescent dye used by the FACS machine) has an excitation and emission profile similar to CFP and was therefore used as a control. A range of concentration of Pacific Blue (diluted in PBS and also in SD medium) was loaded onto a 96 well microtitre plate and analysed using the fluorometer. The resulting reading (Fig.1) indicated that the filters in place were able to detect CFP.

In order to check whether pRS414 was expressing CFP, cultures of BY4741 containing pRS414 were prepared using the old/previously used medium and inducer (concentration of 50 and 10µM copper were used) and also using freshly made medium and inducer stocks. The cultures were incubated at 30°C and then samples were analysed using the fluorometer and a mircoscope equipped with CFP filters.

Results

The fluorometer readings indicated a small increase in fluorescence in samples containing Cu2+. However, the readings were still very close to the readings obtained for the background fluorescence of the yeast cells.
The microscope observations indicated that no CFP fluorescence was present in any of the samples prepared.

Conclusion

We conclude from these results that the lack of CFP expression is not due to experimental error and that the fault must lie with one or several of the components making up the pRS414 construct.