Team:TU-Delft/test4

From 2012.igem.org

(Difference between revisions)
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<h1>text text text</h1>
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<h1><span lang=EN-US style='mso-ansi-language:EN-US'>Abstract:<o:p></o:p></span></h1>
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<h2>text text text</h2>
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<h3>text text text</h3>
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<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><i style='mso-bidi-font-style:
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<p> bdjhfvdfjek </p>
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normal'><span lang=EN-US style='mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></i></b></p>
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<h2><span lang=EN-US style='mso-ansi-language:EN-US'>Introduction:<o:p></o:p></span></h2>
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<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Flow <span
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class=SpellE>cytometry</span> is used to measure the response of the mating
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pathway when the receptor is induced by the ligand. Yeast goes into cell cycle
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arrest (sticking in the G1 phase) after induction. After induction we take time
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points where we stain the yeast DNA and use a flow cytometer to measure
+
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fluorescence intensity and correlate it with DNA amount.<o:p></o:p></span></p>
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-
 
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<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>We
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-
calibrate the system by inducing yeast strains with alpha pheromone and
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measuring excitation peaks and distribution and compare it with non-induced
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-
strains. The side scatter is a parameter which is influenced by the cell
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morphology.<o:p></o:p></span></p>
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-
 
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<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>The DNA
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stain <span class=SpellE>Vybrant</span> <span class=SpellE>DyeCycle</span>
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Green has excitation/emission peaks of 519/563 respectively. Filters of the <span
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class=SpellE>Cytek</span> Flow cytometer are 488 nm (Blue) and 561 nm (Yellow).
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We consider peaks obtained by blue excitation.<o:p></o:p></span></p>
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<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0 width="100%"
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style='width:100.0%;mso-cellspacing:0cm;mso-yfti-tbllook:1184;mso-padding-alt:
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0cm 0cm 0cm 0cm'>
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<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
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  <td width=275 valign=top style='width:206.25pt;padding:0cm 0cm 0cm 0cm'>
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  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
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  normal;page-break-after:avoid'><span style='font-size:12.0pt;font-family:
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  "Times New Roman","serif";mso-fareast-font-family:"Times New Roman";
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  mso-fareast-language:NL;mso-no-proof:yes'><!--[if gte vml 1]><v:shapetype
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  id="_x0000_t75" coordsize="21600,21600" o:spt="75" o:preferrelative="t"
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  path="m@4@5l@4@11@9@11@9@5xe" filled="f" stroked="f">
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  <v:stroke joinstyle="miter"/>
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  <v:formulas>
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    <v:f eqn="if lineDrawn pixelLineWidth 0"/>
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    <v:f eqn="sum @0 1 0"/>
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    <v:f eqn="sum 0 0 @1"/>
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    <v:f eqn="prod @2 1 2"/>
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    <v:f eqn="sum @0 0 1"/>
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    <v:f eqn="prod @6 1 2"/>
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    <v:f eqn="prod @7 21600 pixelWidth"/>
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    <v:f eqn="sum @8 21600 0"/>
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    <v:f eqn="prod @7 21600 pixelHeight"/>
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    <v:f eqn="sum @10 21600 0"/>
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  </v:formulas>
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  <v:path o:extrusionok="f" gradientshapeok="t" o:connecttype="rect"/>
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  <o:lock v:ext="edit" aspectratio="t"/>
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  </v:shapetype><v:shape id="_x002f_etc_x002f_medialib_x002f_en_x002f_images_x002f_ics_organized_x002f_References_x002f_Protocols_x002f_data-chart_x002f_275_x0023_Par.68154.Image_x0020_"
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  o:spid="_x0000_i1029" type="#_x0000_t75" alt="Description: Fluorescence excitation and emission spectra for the Vybrant® DyeCycle™ Orange stain"
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  style='width:206.25pt;height:180.75pt;visibility:visible;mso-wrap-style:square'>
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  <v:imagedata src="analysis_files/image001.gif" o:title="Fluorescence excitation and emission spectra for the Vybrant® DyeCycle™ Orange stain"/>
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-
  </v:shape><![endif]--><![if !vml]><img width=275 height=241
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  src="analysis_files/image001.gif"
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-
  alt="Description: Fluorescence excitation and emission spectra for the Vybrant® DyeCycle™ Orange stain"
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-
  v:shapes="_x002f_etc_x002f_medialib_x002f_en_x002f_images_x002f_ics_organized_x002f_References_x002f_Protocols_x002f_data-chart_x002f_275_x0023_Par.68154.Image_x0020_"><![endif]></span></p>
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  <p class=MsoCaption><span lang=EN-US style='mso-ansi-language:EN-US'>Figure </span><!--[if supportFields]><span
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-
  style='mso-element:field-begin'></span><span lang=EN-US style='mso-ansi-language:
+
-
  EN-US'><span style='mso-spacerun:yes'> </span>SEQ Figure \* ARABIC </span><span
+
-
  style='mso-element:field-separator'></span><![endif]--><span lang=EN-US
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-
  style='mso-ansi-language:EN-US;mso-no-proof:yes'>1</span><!--[if supportFields]><span
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-
  style='mso-element:field-end'></span><![endif]--><span style='mso-ansi-language:
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-
  EN-US'> </span><span lang=EN-US style='mso-ansi-language:EN-US;font-weight:
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  normal'>Fluorescence excitation and emission spectra for the <span
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  class=SpellE>Vybrant</span>® <span class=SpellE>DyeCycle</span>™ Orange stain
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  bound to DNA in <span class=SpellE>TBE<span class=GramE>,pH</span></span>
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  8.3. Source: </span><a
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-
  href="http://www.invitrogen.com/site/us/en/home/References/protocols/cell-and-tissue-analysis/flow-cytometry-protocol/cell-cycle-analysis/vybrant-dyecycle-green-and-orange-stains.html"><span
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-
  lang=EN-US style='color:#4F81BD;mso-themecolor:accent1;mso-ansi-language:
+
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  EN-US;font-weight:normal;text-decoration:none;text-underline:none'>http://www.invitrogen.com/site/us/en/home/References/protocols/cell-and-tissue-analysis/flow-cytometry-protocol/cell-cycle-analysis/vybrant-dyecycle-green-and-orange-stains.html</span></a><span
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-
  lang=EN-US style='font-size:12.0pt;font-family:"Times New Roman","serif";
+
-
  mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US;mso-fareast-language:
+
-
  NL'><o:p></o:p></span></p>
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-
  </td>
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-
</tr>
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-
<tr style='mso-yfti-irow:1;mso-yfti-lastrow:yes'>
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  <td width=275 valign=top style='width:206.25pt;padding:0cm 0cm 0cm 0cm'></td>
+
-
</tr>
+
-
</table>
+
-
 
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-
<h2><span lang=EN-US style='mso-ansi-language:EN-US'>Methods:<o:p></o:p></span></h2>
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-
 
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<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>A frozen
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-
stock is grown overnight on 30 °C and diluted until an OD<sub>600</sub> of
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-
approximately 0.05 was measured. Cells were grown in DO –<span class=SpellE>Leucine</span>
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-
media. Cells transformed with nicotinic acid receptor where grown in DO –<span
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-
class=SpellE>Leucine</span> –Nicotinic acid media. <span class=GramE>Cells
+
-
where centrifuged (10 min 4000 RPM) and media was refreshed.</span> Cells were
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grown for seven hours and centrifuged again. Pellet size is estimated and
+
-
evened. Media was refreshed again. After 2.5 hours of growth (cells in
+
-
exponential phase) cells were induced with ligand. Half an hour before every
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-
time point 1 ml cells is taken and 2 <span class=SpellE>&#956;l</span> <span
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-
class=SpellE>Vybrant</span>® <span class=SpellE>DyeCycle</span>™ Orange is
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-
added, <span class=SpellE>vortexed</span> and kept on 37 °C for half an hour
+
-
and then measured with a <span class=SpellE>Cytek</span> <span class=SpellE>FACScan</span>.
+
-
Graphs were analyzed with <span class=SpellE>Flowjo</span>.<o:p></o:p></span></p>
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-
 
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<h2><span lang=EN-US style='mso-ansi-language:EN-US'>Results<o:p></o:p></span></h2>
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-
 
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<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>We take S. <span
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-
class=SpellE>cerevisae</span> with I7-OR1G1 expression as an example, Time points
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-
are taken at ~T=1 hour, ~T=3.20 hours and T=4.30 hours after induction with
+
-
&#945; pheromone. Figure 2 shows graphs of side scatter versus intensity and a
+
-
histogram of cell intensity of the whole population (N=20.000 cells). <o:p></o:p></span></p>
+
-
 
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-
<p class=MsoNormal><i style='mso-bidi-font-style:normal'><span lang=EN-US
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-
style='mso-ansi-language:EN-US'>Positive control<o:p></o:p></span></i></p>
+
-
 
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<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>A secondary
+
-
cloud can be seen to emerge in the pheromone induced <span class=GramE>population
+
-
which show</span> a reduced side scatter (lower cloud centre). The higher
+
-
intensity indicates an increased amount of DNA.<i style='mso-bidi-font-style:
+
-
normal'><o:p></o:p></i></span></p>
+
-
 
+
-
<p class=MsoNormal style='page-break-after:avoid'><span style='mso-fareast-language:
+
-
NL;mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Afbeelding_x0020_5"
+
-
o:spid="_x0000_i1028" type="#_x0000_t75" alt="Description: Flow_cytometer_OR1G1_alpha_results.png"
+
-
style='width:347.25pt;height:473.25pt;visibility:visible;mso-wrap-style:square'>
+
-
<v:imagedata src="analysis_files/image002.png" o:title="Flow_cytometer_OR1G1_alpha_results"/>
+
-
</v:shape><![endif]--><![if !vml]><img border=0 width=463 height=631
+
-
src="analysis_files/image003.gif"
+
-
alt="Description: Flow_cytometer_OR1G1_alpha_results.png" v:shapes="Afbeelding_x0020_5"><![endif]></span></p>
+
-
 
+
-
<p class=MsoCaption><span lang=EN-US style='mso-ansi-language:EN-US'>Figure </span><!--[if supportFields]><span
+
-
style='mso-element:field-begin'></span><span lang=EN-US style='mso-ansi-language:
+
-
EN-US'><span style='mso-spacerun:yes'> </span>SEQ Figure \* ARABIC </span><span
+
-
style='mso-element:field-separator'></span><![endif]--><span lang=EN-US
+
-
style='mso-ansi-language:EN-US;mso-no-proof:yes'>2</span><!--[if supportFields]><span
+
-
style='mso-element:field-end'></span><![endif]--><span lang=EN-US
+
-
style='mso-ansi-language:EN-US'> Cell intensity distribution of alpha pheromone
+
-
induced S. <span class=SpellE>cerevisae</span> cells. The cells have been
+
-
transformed with I7-Olfr154. Every time point shows <span class=GramE>an
+
-
intensity distribution (top) of (Blue 590-20) excited cells</span>. The lower
+
-
graph shows a SSC versus intensity distribution.<o:p></o:p></span></p>
+
-
 
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-
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
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-
 
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<h2><span lang=EN-US style='mso-ansi-language:EN-US'>Induction of Methyl <span
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-
class=SpellE>nicotinate</span> receptor<o:p></o:p></span></h2>
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-
 
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-
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Figure 3
+
-
shows results cells transformed with I7-Gpr109A were induced by concentrations
+
-
of methyl <span class=SpellE>nicotinate</span>. <span class=GramE>The negative
+
-
control, non-induced cells over time, show two clouds relatively close to each
+
-
other, similar to the negative control of the alpha induced cells.</span> Cells
+
-
induced with methyl <span class=SpellE>nicotinate</span> show very low
+
-
alteration of intensity, only at t=3.25 hours a slight cloud shift towards the
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-
10<sup>3</sup> intensity level can be observed. Cells induced with nicotinic
+
-
acid show a very broad shift from an intensity of 10<sup>4</sup> to an intensity
+
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of 10<sup>3</sup>. This indicates that the DNA content per cell dropped. The
+
-
specific negative control without receptor but with a high nicotinic acid
+
-
receptor also has a drop in DNA content. This indicates that a high nicotinic
+
-
acid can be the cause of the drop of DNA content, for example through increased
+
-
cell division. Therefore, vitamin addition<o:p></o:p></span></p>
+
-
 
+
-
<p class=MsoNormal style='page-break-after:avoid'><span style='mso-fareast-language:
+
-
NL;mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Afbeelding_x0020_1"
+
-
o:spid="_x0000_i1027" type="#_x0000_t75" alt="Description: C:\Users\Mark\Pictures\Flow_cytometer_Gpr109A_results.png"
+
-
style='width:453.75pt;height:482.25pt;visibility:visible;mso-wrap-style:square'>
+
-
<v:imagedata src="analysis_files/image004.png" o:title="Flow_cytometer_Gpr109A_results"/>
+
-
</v:shape><![endif]--><![if !vml]><img border=0 width=605 height=643
+
-
src="analysis_files/image005.gif"
+
-
alt="Description: C:\Users\Mark\Pictures\Flow_cytometer_Gpr109A_results.png"
+
-
v:shapes="Afbeelding_x0020_1"><![endif]></span></p>
+
-
 
+
-
<p class=MsoCaption><span lang=EN-US style='mso-ansi-language:EN-US'>Figure </span><!--[if supportFields]><span
+
-
style='mso-element:field-begin'></span><span lang=EN-US style='mso-ansi-language:
+
-
EN-US'><span style='mso-spacerun:yes'> </span>SEQ Figure \* ARABIC </span><span
+
-
style='mso-element:field-separator'></span><![endif]--><span lang=EN-US
+
-
style='mso-ansi-language:EN-US;mso-no-proof:yes'>3</span><!--[if supportFields]><span
+
-
style='mso-element:field-end'></span><![endif]--><span style='mso-ansi-language:
+
-
EN-US'> <span lang=EN-US>Cell intensity distribution of ligand induced S. <span
+
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class=SpellE>cerevisae</span> cells. The cells have been transformed with
+
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I7-Gpr109A. Every time point shows <span class=GramE>an intensity distribution
+
-
(top) of (Blue 590-20) excited cells</span>. The lower graph shows a SSC versus
+
-
intensity distribution.<o:p></o:p></span></span></p>
+
-
 
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<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
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<h2><span lang=EN-US style='mso-ansi-language:EN-US'>Induction of Banana smell
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(<span class=SpellE>isoamyl</span> acetate) receptor<o:p></o:p></span></h2>
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-
 
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<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Figure 4
+
-
shows results of cells transformed with I7-Olfr154, I7-OR1G1 and <span
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class=SpellE>wildtype</span> cells were induced by concentrations of <span
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class=SpellE>isoamyl</span> acetate. The negative control, non-induced cells
+
-
over time (first and fourth column), show two clouds relatively close to each
+
-
other, similar to the negative control of the alpha induced cells. The same
+
-
holds for the <span class=SpellE>wildtype</span> cells (third column). The
+
-
I7-Olfr154 transformed cells (second column) shows at T=1.20 a slight increase
+
-
in cell intensity, combined with a low SSC. At time points t=3.08 and t=4.30
+
-
this cannot be observed and the data shows little deviation from the negative
+
-
control. The I7-OR1G1 transformed cells however, maintain this deviation in <span
+
-
style='background:red;mso-highlight:red'>all three time points</span>. There
+
-
can also be noticed that this deviation is towards the 10<sup>5</sup> region
+
-
instead of 10<sup>3</sup> with the nicotinic acid induced cells, similar to the
+
-
secondary cloud of the alpha pheromone induced cells.<i style='mso-bidi-font-style:
+
-
normal'><o:p></o:p></i></span></p>
+
-
 
+
-
<p class=MsoNormal style='page-break-after:avoid'><i style='mso-bidi-font-style:
+
-
normal'><span style='mso-fareast-language:NL;mso-no-proof:yes'><!--[if gte vml 1]><v:shape
+
-
id="Afbeelding_x0020_7" o:spid="_x0000_i1026" type="#_x0000_t75" alt="Description: Flow_cytometer_OR1G1_IsoAmyl_Results_NOTDONE.png"
+
-
style='width:490.5pt;height:449.25pt;visibility:visible;mso-wrap-style:square'>
+
-
<v:imagedata src="analysis_files/image006.png" o:title="Flow_cytometer_OR1G1_IsoAmyl_Results_NOTDONE"/>
+
-
</v:shape><![endif]--><![if !vml]><img border=0 width=654 height=599
+
-
src="analysis_files/image007.gif"
+
-
alt="Description: Flow_cytometer_OR1G1_IsoAmyl_Results_NOTDONE.png" v:shapes="Afbeelding_x0020_7"><![endif]></span></i></p>
+
-
 
+
-
<p class=MsoCaption><span lang=EN-US style='mso-ansi-language:EN-US'>Figure </span><!--[if supportFields]><span
+
-
style='mso-element:field-begin'></span><span lang=EN-US style='mso-ansi-language:
+
-
EN-US'><span style='mso-spacerun:yes'> </span>SEQ Figure \* ARABIC </span><span
+
-
style='mso-element:field-separator'></span><![endif]--><span lang=EN-US
+
-
style='mso-ansi-language:EN-US;mso-no-proof:yes'>4</span><!--[if supportFields]><span
+
-
style='mso-element:field-end'></span><![endif]--><span style='mso-ansi-language:
+
-
EN-US'> <span lang=EN-US>Cell intensity distribution of ligand induced S. <span
+
-
class=SpellE>cerevisae</span> cells. The cells have been transformed with
+
-
I7-Olfr154 and I7-OR1G1. Every time point shows <span class=GramE>an intensity
+
-
distribution (top) of (Blue 590-20) excited cells</span>. The lower graph shows
+
-
a SSC versus intensity distribution.<o:p></o:p></span></span></p>
+
-
 
+
-
<p class=MsoCaption><i style='mso-bidi-font-style:normal'><span lang=EN-US
+
-
style='mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></i></p>
+
-
 
+
-
<h2><span lang=EN-US style='mso-ansi-language:EN-US'>Induction of <span
+
-
class=SpellE>diacetyl</span> receptor<o:p></o:p></span></h2>
+
-
 
+
-
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Figure 3
+
-
shows results of cells transformed with I7-Odr10 were induced by concentrations
+
-
of 2<span class=GramE>,3</span> <span class=SpellE>butadione</span> (<span
+
-
class=SpellE>diacetyl</span>). <span class=GramE>The negative control,
+
-
non-induced cells over time show a deviation towards the 10<sup>3</sup> region.</span>
+
-
The gain has been lowered between T=1.06 and T=3.18 points since high intensity
+
-
points ‘dropped off’. No significant difference between the induced and
+
-
non-induced data can be seen. What can be noted is that no secondary region can
+
-
be detected, which can be seen for <span class=SpellE>wiltype</span> induced
+
-
with <span class=SpellE>diacetyl</span>.</span><i style='mso-bidi-font-style:
+
-
normal'><span style='mso-fareast-language:NL;mso-no-proof:yes'><!--[if gte vml 1]><v:shape
+
-
id="Afbeelding_x0020_12" o:spid="_x0000_i1025" type="#_x0000_t75" alt="Description: Flow_cytometer_Diacetyl.png"
+
-
style='width:453.75pt;height:480.75pt;visibility:visible;mso-wrap-style:square'>
+
-
<v:imagedata src="analysis_files/image008.png" o:title="Flow_cytometer_Diacetyl"/>
+
-
</v:shape><![endif]--><![if !vml]><img border=0 width=605 height=641
+
-
src="analysis_files/image009.gif" alt="Description: Flow_cytometer_Diacetyl.png"
+
-
v:shapes="Afbeelding_x0020_12"><![endif]></span></i><i style='mso-bidi-font-style:
+
-
normal'><span lang=EN-US style='mso-ansi-language:EN-US'><o:p></o:p></span></i></p>
+
   
   

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