Confirming the CFP sequence is functional

Aim

As part of the experiments to repair the pRS414 construct we need to determine if the CFP sequence used is functional or not. By confirming that the CFP protein is functional we can narrow down where the fault lies in the construct. If the CFP sequence was not functional this could mean that the construct was expressing properly but we simply could not detect this.

Hypothesis

The CFP sequence is accurate and the reason why no expression of CFP is being observed is because the protein is not being synthesised properly or not all.

Protocol

The YCp lac 22 Fl plasmid has been used to drive the expression of fluorescent proteins. The TEF1 promoter has been shown to constitutively express GFP at high levels.

A restriction digest was performed using the enzymes Nde1 and Xba1 in order to remove GFP from the construct. The CFP sequence in pRS414 was then PCR amplified using primers to generate complementary overhangs to allow homologous recombination into the gaped Ycp lac 22 Fl vector.

The gapped vector and the PCR product were co-transformed into yeast (BY4741ΔTrp strain) and incubated for several days. The resulting transformants were then cultured overnight in SD medium. Sample were washed and re-suspended in PBS and then observed under a microscope fitted with CFP filters.

Results

We can see from Fig.3 that the CFP protein is being properly expressed. This means that the sequence used for CFP in the pRS414 construct is accurate and should be working. During this experiment the sequence of MS2-CFP was also checked to ensure that during the design both proteins had remained in frame.

We can conclude from this experiment that the reason we are not seeing any CFP expression in pRS414 is because the CFP protein is not being produced properly.