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3. N-GFP Swap Experiment
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Revision as of 11:35, 26 October 2010
University of Aberdeen - ayeSwitch
Replacement of MS2-[CFP] in CUP1p - [MS2-CFP] with Npep-GFP from GAL1p-[Npep-GFP] to Determine Functionality of Promoter/5'leader/Binding Stem Loop Sequence
Aim
The aim of this experiment is to see if the CUP1p - [MS2-CFP] 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 GAL1p-[Npep-GFP]. We have shown that Npep-GFP is expressed by GAL1p-[Npep-GFP] therefore if we can express it in CUP1p - [MS2-CFP] we can determine the functionality of the promoter/5'leader/binding stem loop sequence.
Hypothesis
The reason why CUP1p - [MS2-CFP] is not expressing CFP lies within the promoter/5’leader/stem loop sequence.
Protocol
The CUP1p - [MS2-CFP] construct was digested using the restriction enzymes Cla1 and Nde1 in order to remove the MS2-CFP section of CUP1p - [MS2-CFP].
In parallel, the Npeptide-GFP section of GAL1p-[Npep-GFP] 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 CUP1p - [MS2-CFP]. The gapped CUP1p - [MS2-CFP] 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 “GAL1p-[Npep-GFP]”. 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 CUP1p - [MS2-CFP] construct is still not expressing properly.
Conclusion
We can conclude from these results that the problem resulting in CUP1p - [MS2-CFP] 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.
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