4. Replacing the CUP1 promoter in pRS414 with the CUP1-2 promoter from the N4 construct

From 2010.igem.org

University of Aberdeen - ayeSwitch - iGEM 2010

Checking Copper Promoter in CUP1p - [MS2-CFP] by Replacing it with CUP1-2 Promoter from CUP1p-[GFP]Construct


When comparing the sequence of the promoter present in CUP1p - [MS2-CFP] to the sequence of the promoter in CUP1p-[GFP] we noticed that the CUP1p-[GFP] sequence contained 50 base pairs in its associated 5’UTR that were not present in the CUP1p - [MS2-CFP] 5’UTR sequence. We have shown that the CUP1p-[GFP] promoter works (Characterisation of Cup1 Promoter experiments). By replacing the promoter in CUP1p - [MS2-CFP] with the promoter and associated 5’UTR from CUP1p-[GFP] we can determine whether or not CUP1p - [MS2-CFP] had a defective or incomplete promoter which resulted in no expression of CFP.


The Cup promoter present in CUP1p-[GFP] contains fifty base pairs in its associated 5’UTR that are not present in the CUP1p - [MS2-CFP] construct which are responsible for the pRS414 construct not expressing CFP properly.


The CUP1p - [MS2-CFP] construct was digested using the restriction enzymes Bgl2 and Pst1 in order to remove the existing Cup1 promoter. The promoter present in CUP1p-[GFP] (Cup1-2) and the associated 5’UTR were then PCR amplified using primers designed to add complementary overhangs to the gapped CUP1p - [MS2-CFP] construct to allow homologous recombination.

The gapped CUP1p - [MS2-CFP] vector and the PCR amplified CUP1-2 promoter were then co-transformed into yeast (BY4741ΔTrp strain) and incubated over several days. The resulting transformants were cultured in SD medium containing CuSO4 at concentrations high enough to reach full induction of the promoter. Final samples were washed and re-suspended in PBS and then analysed using a microscope fitted with CFP filters.


Microscope analysis revealed no fluorescence coming from the cell samples under the CFP excitation and filter settings. Controls containing pGAL/GFP showed clear fluorescence however only background fluorescence from the yeast cells was observed coming from the cells containing Cup1/CFP. 20 different samples were tested using different colonies form the transformation plate each time however all results came up negative. As we know that the promoter repaired into the CUP1p - [MS2-CFP] construct worked (see Characterisation of Cup1 Promoter experiments) we can conclude that the initial lack of CFP expression observed was not due to a faulty promoter but must stem from either the Bbox stem loop or the fusion of MS2 to CFP.

Back to the Top