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

From 2010.igem.org

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<p>The Cup promoter present in N4 contains fifty base pairs in its associated 5’UTR that are not present in the pRS414 construct which are responsible for the pRS414 construct not expressing CFP properly.</p>
<p>The Cup promoter present in N4 contains fifty base pairs in its associated 5’UTR that are not present in the pRS414 construct which are responsible for the pRS414 construct not expressing CFP properly.</p>
<h3>Protocol</h3>
<h3>Protocol</h3>
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<p>The pRS414 construct was digested using the restriction enzymes Bgl2 and Pst1 in order to remove the existing Cup1 promoter. The promoter present in N4 (Cup1-2) and the associated 5’UTR were then PCR amplified using primers designed to add complementary overhangs to the gapped pRS414 construct to allow homologous recombination.  
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<p>The pRS414 construct was digested using the restriction enzymes Bgl2 and Pst1 in order to remove the existing Cup1 promoter. The promoter present in N4 (Cup1-2) and the associated 5’UTR were then PCR amplified using primers designed to add complementary overhangs to the gapped pRS414 construct to allow homologous recombination.</p>
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<img src="http://2010.igem.org/wiki/images/8/81/PRS414_construct.jpg"/>
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<br>The gapped pRS414 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 were then analysed using a microscope fitted with CFP filters.<br>
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<br><p>The gapped pRS414 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 were then analysed using a microscope fitted with CFP filters.<br>
<h3>Results</h3>
<h3>Results</h3>
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<img src="http://2010.igem.org/wiki/images/0/0a/Absence_of_CFP_fluoresence.jpg"/>
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<p>Microscope analysis revealed no fluorescence coming form 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 pRS414 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.  
<p>Microscope analysis revealed no fluorescence coming form 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 pRS414 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.  
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Revision as of 18:25, 8 October 2010

University of Aberdeen - ayeSwitch - iGEM 2010

Checking the copper promoter in pRS414 by replacing it with the CUP1-2 promoter from the N4 construct

Aim

When comparing the sequence of the promoter present in pRS414 to the sequence of the promoter in N4 we noticed that the N4 sequence contained 50 base pairs in its associated 5’UTR that were not present in the pRS414 5’UTR sequence. We have shown that the N4 promoter works (Characterisation of Cup1 Promoter experiments). By replacing the promoter in pRS414 with the promoter and associated 5’UTR from N4 we can determine whether or not pRS414 had a defective or incomplete promoter which resulted in no expression of CFP.

Hypothesis

The Cup promoter present in N4 contains fifty base pairs in its associated 5’UTR that are not present in the pRS414 construct which are responsible for the pRS414 construct not expressing CFP properly.

Protocol

The pRS414 construct was digested using the restriction enzymes Bgl2 and Pst1 in order to remove the existing Cup1 promoter. The promoter present in N4 (Cup1-2) and the associated 5’UTR were then PCR amplified using primers designed to add complementary overhangs to the gapped pRS414 construct to allow homologous recombination.


The gapped pRS414 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 were then analysed using a microscope fitted with CFP filters.

Results

Microscope analysis revealed no fluorescence coming form 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 pRS414 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.

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