Copper Dose Response of the Cup1 promoter using N4

From 2010.igem.org

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<h1>Quantitative derermination of the response of the CUP1 promoter to varying concentration of CuSO4</h1>
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<h3>Aim<h3>
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<p>The aim of this experiment is to characterise the CUP1 promoter present on N4 by determining whether
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it displays a dose response quality. The determined characteristics of this promoter can then be applied to the promoter present in pRS414 in order to allow more precise modelling of the switch.</p>
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<h3>Hypothesis</h3>
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<p>The CUP1 promoter ixhibits a linera relationship between the concentration of CuSO4 and the level of GFP expression when CuSO4 concentrations range from 0microM to 100microM.  At concentrations of 100microM and hicher the expression level of GFP will have reached a steady state and will remain unchanged despite increasing CuSO4 concetrations. [1]</p>
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<h3>Protocol</h3>
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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.
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<p class=MsoNormalCxSpFirst style='text-indent:36.0pt'><span style='font-size:
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18.0pt;line-height:115%'>Results</span></p>
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<p class=MsoNormalCxSpMiddle>&nbsp;</p>
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<p class=MsoNormalCxSpMiddle><b><u><span style='font-size:12.0pt;line-height:
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115%'>Title:</span></u></b><b><span style='font-size:12.0pt;line-height:115%'> </span></b></p>
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<p class=MsoNormalCxSpMiddle align=center style='text-align:center'><u><span
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style='font-size:14.0pt;line-height:115%'>Quantitative determination of the
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response of the CUP1 promoter to varying concentrations of CuSo4</span></u></p>
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<p class=MsoNormalCxSpMiddle>&nbsp;</p>
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<p class=MsoNormalCxSpMiddle><b><u><span style='font-size:12.0pt;line-height:
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115%'>Aim</span></u></b></p>
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<p class=MsoNormalCxSpMiddle style='text-indent:36.0pt'>The aim of this
+
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experiment is to characterise the CUP1 promoter present on N4 by determining
+
-
whether it displays a dose response quality. The determined characteristics of
+
-
this promoter can then be applied to the promoter present in pRS414 in order to
+
-
allow more precise modelling of the switch.</p>
+
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+
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<p class=MsoNormalCxSpMiddle style='text-indent:36.0pt'>&nbsp;</p>
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<p class=MsoNormalCxSpMiddle><b><u><span style='font-size:12.0pt;line-height:
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115%'>Hypothesis </span></u></b></p>
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<p class=MsoNormalCxSpMiddle style='text-indent:36.0pt'>The CUP1 promoter
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exhibits a linear relationship between [CuSO4] and the level of GFP expression
+
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when CuSO4 concentrations range from 0µM to 100µM. At concentrations of 100µM
+
-
and higher the expression level of GFP will have reached a steady state and
+
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will remain unchanged despite increasing CuSO4 concentrations. [1]</p>
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<p class=MsoNormalCxSpMiddle>&nbsp;</p>
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<p class=MsoNormalCxSpMiddle><b><u><span style='font-size:12.0pt;line-height:
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115%'>Protocol</span></u></b></p>
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<p class=MsoNormalCxSpMiddle style='text-indent:36.0pt'>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.</p>
+
-
 
+
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<p class=MsoNormalCxSpMiddle style='text-indent:36.0pt'>Three separate starter
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cultures were prepared of N4 in 5mL SD medium. The following tubes were then
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prepared in triplicate</p>
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<p class=MsoNormalCxSpMiddle>&nbsp;</p>
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  style='color:black'>Tube n°</span></b></p>
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  style='color:black'>1</span></b></p>
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  style='color:black'>2</span></b></p>
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  style='color:black'>3</span></b></p>
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  style='color:black'>4</span></b></p>
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  style='color:black'>5</span></b></p>
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  style='color:black'>6</span></b></p>
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  style='color:black'>7</span></b></p>
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  style='color:black'>8</span></b></p>
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  margin-bottom:.0001pt;text-align:center;line-height:normal'><b><span
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  style='color:black'>[CuSO4] (µM)</span></b></p>
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  style='color:black'>0</span></p>
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  margin-bottom:.0001pt;text-align:center;line-height:normal'><span
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  style='color:black'>10</span></p>
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  style='color:black'>15</span></p>
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  style='color:black'>20</span></p>
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  style='color:black'>25</span></p>
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  style='color:black'>50</span></p>
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  style='color:black'>75</span></p>
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  style='color:black'>100</span></p>
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<p class=MsoNormalCxSpMiddle style='text-indent:36.0pt'>Each one of these tubes
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was inoculated with N4 from each starter culture in order to provide
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triplicates of each concentration value. The cells were later harvested once
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the OD<span style='font-size:8.0pt;line-height:115%'>600</span> had reached 0.6.
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Triplicates from each culture were then loaded onto a 96 microtitre plate. The
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fluorescence of each sample was measured using a fluoremeter running the
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“RussGFP” protocol.</p>
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<p class=MsoNormalCxSpMiddle style='text-indent:36.0pt'>&nbsp;</p>
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<p class=MsoNormalCxSpMiddle><b><u><span style='font-size:12.0pt;line-height:
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115%'>Result</span></u></b></p>
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<p class=MsoNormalCxSpMiddle style='text-indent:36.0pt'>For full data
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spreadsheet</p>
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<p class=MsoNormalCxSpMiddle><span style='font-family:Wingdings'>è</span>IGEM
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240610 JH+SL PCup Induction.xslx</p>
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    <p class=MsoCaption align=center style='text-align:center'>Figure 1 Plot showing
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    the relationship between the average GFP expression and [CuSO4]</p>
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<p class=MsoNormalCxSpMiddle><b><u><span style='font-size:12.0pt;line-height:
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<p class=MsoNormalCxSpMiddle style='text-indent:36.0pt'>The data suggests
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<p class=MsoNormalCxSpMiddle>[1]. Gorman JA, Clark PE, Lee MC, Debouck C and
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Rosenberg M</p>
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<p class=MsoNormalCxSpMiddle style='text-indent:36.0pt'>Regulation of the yeast
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metallothionein gene</p>
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<b>Return to [[https://2010.igem.org/Team:Aberdeen_Scotland/Results]] page</b>
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Three separate starter cultures were prepared of N4 in 5mL SD medium. the following tubes were then prepared in triplicate.</p>

Revision as of 13:43, 8 October 2010

University of Aberdeen - ayeSwitch - iGEM 2010

Quantitative derermination of the response of the CUP1 promoter to varying concentration of CuSO4

Aim

The aim of this experiment is to characterise the CUP1 promoter present on N4 by determining whether it displays a dose response quality. The determined characteristics of this promoter can then be applied to the promoter present in pRS414 in order to allow more precise modelling of the switch.

Hypothesis

The CUP1 promoter ixhibits a linera relationship between the concentration of CuSO4 and the level of GFP expression when CuSO4 concentrations range from 0microM to 100microM. At concentrations of 100microM and hicher the expression level of GFP will have reached a steady state and will remain unchanged despite increasing CuSO4 concetrations. [1]

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.
Three separate starter cultures were prepared of N4 in 5mL SD medium. the following tubes were then prepared in triplicate.