Timed Induction of the Cup1 promoter using N4

From 2010.igem.org

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<h1>Confirmation using microscope and fluorometer analysis that pRS414 was not expressing CFP</h1>
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<h1>Timed induction of GFP expression by the CUP1 promoter</h1>
<h3>Aim</h3>
<h3>Aim</h3>
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<p>The aim of this experiment is to determine whether the pRS414 construct can express CFP in the presence of CuSO4.  The detection of CFP using the fluorometer and the microscope would confirm whether the construct as a whole was functional or not.</p>
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<p>The aim of this experiment is to determine how quickly the Cup1 promoter is fully induced by set concentrations of copper by measuring the levels of GFP being expressed by the cells.</p>
<h3>Hypothesis</h3>
<h3>Hypothesis</h3>
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<p>The observed lack of CFP expression is due to experimental errors and in appropriate medium and the presence of proper inducing agent, the pRS414 construct will express CFP.</p>
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<p>The CUP1 promoter will rapidly become fully induced following exposure to set concentrations of copper.  Once it is fully induced the level of GFP expression will stabilise and will no longer increase despite more time passing.</p>
<h3>Protocol</h3>
<h3>Protocol</h3>
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<p>The Cyan filters on the fluorometer were first checked to ensure that they could detect CFP.  Pacific blue beads (a fluorescent dye used by the FACS machine) has and excitation and emission profile similar to CFP and was therefore used as a control.  A range of concentration of Pacific Blue (diluted in PBS and also in SD medium) was loaded onto a 96 well microtitre plate and analysed using the fluoremeter. The resulting reading (Fig.1) indicated that the filters in place were able to detect CFP.
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<p>A genomically integrated GFP gene under control of a CUP1 promoter was used to characterise the control properties of this promoter, this construct is referred to as N4 and was transformed into the yeast strain BY4741 for analysis.<br><br>
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Starter cultures of N4 in 5mL of SD medium + Raffinose were set up and incubated overnight. A 50mL flask containing SD Raff was then inoculated using the starter cultures and incubated overnight until the OD600 reached 0.3.<br><br>
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At t=0 a sample from the flask was put on ice to provide a background reading of yeast’s natural fluorescence without any inducer. Copper was then added to the flask providing a concentration of 100μM. Samples were then taken every 20 minutes and put on ice. All samples were then normalised to an OD600 of 0.75 and were washed and re-suspended in PBS before being analysed in the fluoremeter (the programme RussGFP” was used for the analysis).</p>
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<img src="http://2010.igem.org/wiki/images/d/d8/Table_for_Confirmation_that_PRS414_was_not_expressing_CFP_.png"/>
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</center>
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<br>
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<p>In order to check whether pRS414 was expressing CFP, cultures of BY4741 containing pRS414 were prepared using the old/previously used medium inducer (concentration of 50 and 10microM copper were used) and also using freshly made medium and inducer stocks. The cultures were incubated a 30degreesC and then samples were analysed using the fluorometer and a mircoscope equipped with CFP filters.</p>
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<h3>Results</h3>
<h3>Results</h3>
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For full data spreadsheet<br>
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<img src="http://2010.igem.org/wiki/images/5/57/Table2_for_Confirmation_that_PRS414_was_not_expressing_CFP_.png"/>
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iGEM 2.6.10 JH+SL N4 Timed Induction.xslx<br>
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<br>
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<p>The fluorometer readings indicated a small increase in fluorescence in samples containing Cu2+.  However, the readings were still very close to the readings obtained for the background fluorescence of the yeast cells.
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The microscope observations indicated that no CFP fluorescence was present in any of the samples prepared.</p>
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<br>[JPEG coming soon]<br>
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</p>
<h3>Conclusion</h3>
<h3>Conclusion</h3>
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<p>We conclude from these results that the lack of CFP expression is not due to experimental error and that the fault must lie with one or several of the components making up the pRS414 construct.</p>
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<p>Figure 1 indicates that full induction was reached after approximately 80 minutes and that the level of GFP expression seems to reach a plateau after this and no longer increases. This supports our initial hypothesis.</p>
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<b>Return to [[http://2010.igem.org/Team:Aberdeen_Scotland/Results]] page</b>
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<b>[[http://2010.igem.org/Team:Aberdeen_Scotland/Results Return to Results Main page]]</b>

Revision as of 14:48, 8 October 2010

University of Aberdeen - ayeSwitch - iGEM 2010

Timed induction of GFP expression by the CUP1 promoter

Aim

The aim of this experiment is to determine how quickly the Cup1 promoter is fully induced by set concentrations of copper by measuring the levels of GFP being expressed by the cells.

Hypothesis

The CUP1 promoter will rapidly become fully induced following exposure to set concentrations of copper. Once it is fully induced the level of GFP expression will stabilise and will no longer increase despite more time passing.

Protocol

A genomically integrated GFP gene under control of a CUP1 promoter was used to characterise the control properties of this promoter, this construct is referred to as N4 and was transformed into the yeast strain BY4741 for analysis.

Starter cultures of N4 in 5mL of SD medium + Raffinose were set up and incubated overnight. A 50mL flask containing SD Raff was then inoculated using the starter cultures and incubated overnight until the OD600 reached 0.3.

At t=0 a sample from the flask was put on ice to provide a background reading of yeast’s natural fluorescence without any inducer. Copper was then added to the flask providing a concentration of 100μM. Samples were then taken every 20 minutes and put on ice. All samples were then normalised to an OD600 of 0.75 and were washed and re-suspended in PBS before being analysed in the fluoremeter (the programme RussGFP” was used for the analysis).

Results

For full data spreadsheet
iGEM 2.6.10 JH+SL N4 Timed Induction.xslx


[JPEG coming soon]

Conclusion

Figure 1 indicates that full induction was reached after approximately 80 minutes and that the level of GFP expression seems to reach a plateau after this and no longer increases. This supports our initial hypothesis.

[Return to Results Main page]