N-GFP Swap Experiment

Aim

The aim of this experiment is to see if the pRS414 construct possesses a working promoter/5’leader/binding stem loop sequence by replacing the MS2-CFP part of the construct with the Npep-GFP sequence from pRS415. We have shown that Npep-GFP is expressed by pRS415 therefore if we can express it in pRS414 we can narrow down where the fault is located in pRS414.

Hypothesis

The reason why pRS414 is not expressing CFP lies within the promoter/5’leader/stem loop sequence.

Protocol

The pRS414 construct was digested using the restriction enzymes Cla1 and Nde1 in order to remove the MS2-CFP section of pRS414.

In parallel, the Npeptide-GFP section of pRS415 was PCR amplified using primers designed to add 45 base pair long overhangs that would allow homologous recombination of the PCR product into the gapped pRS414. The gapped pRS414 and the PCR product were co-transformed into yeast (BY4741ΔTrp strain) in order to allow the homologous recombination to take place. The resulting transformants were then screened using Colony PCR in order to make sure that the swap had taken place.

The colonies where the swap had been successful were then cultured overnight in SD medium containing Raffinose and CuSO4 (100µM). Samples were then normalised and resuspended in PBS and loaded onto a 96 well microtitre plate before being analysed using the FACS machine.

Results

From Fig 4. we can determine the level of background fluorescence of yeast cells using the results from the negative control “pRS415”. We can also see the expected levels of GFP in a working construct from the positive control of “pRS415 + 2% Gal”.

The two samples from colony 10, which are in presence of Cu2+ inducer, show readings that correspond with the levels of background fluorescence of yeast cells. This indicates that although the swap was successful the pRS414 construct is still not expressing properly.

Conclusion

We can conclude from these results that the problem resulting in pRS414 not being functional is related to either the Copper promoter or to the 5’UTR and Bbox stem loop. This experiment does not rule out however that the MS2-CFP part of the construct is also defective.