Team:Cambridge/Project/Carotenoid

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<p class=MsoNormal><b><span lang=EN-GB style='font-size:22.0pt;font-family:
 
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Vivaldi'>Colour pigment: Carotenoid</span></b></p>
 
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<p class=MsoNormal><b><span lang=EN-GB style='font-size:16.0pt;font-family:
 
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Vivaldi'>&nbsp;</span></b></p>
 
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<p class=MsoNormal><b><u><span lang=EN-GB style='font-size:18.0pt;font-family:
 
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Vivaldi'>Introduction</span></u></b></p>
 
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<p class=MsoNormal><b><span lang=EN-GB style='font-family:Arial'>Carotenoids</span></b><span
 
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lang=EN-GB style='font-family:Arial'> are organic pigments naturally present in
 
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plants, algae and some bacteria. There are more than 600 carotenoids, which can
 
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be categorised into xanthophylls (hydrocarbons containing oxygen element) and
 
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carotenes (hydrocarbons containing no oxygen). Carotenoids perform a range of
 
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functions, including light energy absorption, protection against photo-damage,
 
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acting as antioxidants, and as precursor to other organic compounds. In human,
 
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for example, beta-carotene is the precursor to vitamin A.</span></p>
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>&nbsp;</span></p>
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>Part of the <b>carotenoid
 
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biosynthesis pathway</b> is shown below (figure adopted from iGEM 2007 Team
 
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Edinburgh):</span></p>
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>&nbsp;</span></p>
 
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[[[Image:Image001.jpg]]]
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>&nbsp;</span></p>
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>Some of the
 
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intermediates in the above pathways are coloured, e.g. lycopene (red),
 
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beta-carotene (orange), and zeaxanthin (yellow). The enzymes involved in the
 
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biosynthetic pathway are <b>CrtE</b>, <b>CrtB</b>, <b>CrtI</b>, <b>CrtY</b>,
 
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and <b>CrtZ</b>. Since our team aims to use different colour pigments as signal
 
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output, the above pathway can be incorporated in our design of colour
 
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production.</span></p>
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>&nbsp;</span></p>
 
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<p class=MsoNormal><b><u><span lang=EN-GB style='font-size:18.0pt;font-family:
 
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Vivaldi'>Research by previous igem teams</span></u></b></p>
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>A few iGEM teams
 
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of previous years (e.g. Edinburgh 07, Edinburgh 08, Guelph 08) conducted
 
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research in the carotenoid system. We are fortunate to have access to some of
 
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their Biobricks in the registry. Here is a summary of the Biobricks available:</span></p>
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>&nbsp;</span></p>
 
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<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
 
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style='border-collapse:collapse;border:none'>
 
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<tr>
 
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  <td width=80 valign=top style='width:60.3pt;border:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><b><span lang=EN-GB style='font-family:Arial'>Registry
 
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  code</span></b></p>
 
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  </td>
 
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  <td width=116 valign=top style='width:86.85pt;border:solid windowtext 1.0pt;
 
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  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><b><span lang=EN-GB style='font-family:Arial'>Team</span></b></p>
 
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  </td>
 
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  <td width=213 valign=top style='width:159.55pt;border:solid windowtext 1.0pt;
 
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  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><b><span lang=EN-GB style='font-family:Arial'>Sequence Description</span></b></p>
 
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  </td>
 
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  <td width=181 valign=top style='width:136.1pt;border:solid windowtext 1.0pt;
 
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  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><b><span lang=EN-GB style='font-family:Arial'>Note</span></b></p>
 
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  </td>
 
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</tr>
 
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<tr>
 
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  <td width=80 valign=top style='width:60.3pt;border:solid windowtext 1.0pt;
 
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  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>K118014</span></p>
 
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  </td>
 
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  <td width=116 valign=top style='width:86.85pt;border-top:none;border-left:
 
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  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>Edinburgh 08</span></p>
 
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  </td>
 
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  <td width=213 valign=top style='width:159.55pt;border-top:none;border-left:
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>rbs + CrtE</span></p>
 
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  </td>
 
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  <td width=181 valign=top style='width:136.1pt;border-top:none;border-left:
 
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  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>&nbsp;</span></p>
 
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  </td>
 
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</tr>
 
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<tr>
 
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  <td width=80 valign=top style='width:60.3pt;border:solid windowtext 1.0pt;
 
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  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>K118006</span></p>
 
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  </td>
 
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  <td width=116 valign=top style='width:86.85pt;border-top:none;border-left:
 
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  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>Edinburgh 08</span></p>
 
-
  </td>
 
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  <td width=213 valign=top style='width:159.55pt;border-top:none;border-left:
 
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  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>rbs + CrtB</span></p>
 
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  </td>
 
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  <td width=181 valign=top style='width:136.1pt;border-top:none;border-left:
 
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  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>&nbsp;</span></p>
 
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  </td>
 
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</tr>
 
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<tr>
 
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  <td width=80 valign=top style='width:60.3pt;border:solid windowtext 1.0pt;
 
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  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>K118005</span></p>
 
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  </td>
 
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  <td width=116 valign=top style='width:86.85pt;border-top:none;border-left:
 
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  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>Edinburgh 08</span></p>
 
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  </td>
 
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  <td width=213 valign=top style='width:159.55pt;border-top:none;border-left:
 
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  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>rbs + CrtI</span></p>
 
-
 
-
  </td>
 
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  <td width=181 valign=top style='width:136.1pt;border-top:none;border-left:
 
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  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>2 PstI sites
 
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  removed.</span></p>
 
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  </td>
 
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</tr>
 
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<tr>
 
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  <td width=80 valign=top style='width:60.3pt;border:solid windowtext 1.0pt;
 
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  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>K118013</span></p>
 
-
 
-
  </td>
 
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  <td width=116 valign=top style='width:86.85pt;border-top:none;border-left:
 
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  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>Edinburgh 08</span></p>
 
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  </td>
 
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  <td width=213 valign=top style='width:159.55pt;border-top:none;border-left:
 
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  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>rbs + CrtY</span></p>
 
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  </td>
 
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  <td width=181 valign=top style='width:136.1pt;border-top:none;border-left:
 
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  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
-
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>&nbsp;</span></p>
 
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  </td>
 
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</tr>
 
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<tr>
 
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  <td width=80 valign=top style='width:60.3pt;border:solid windowtext 1.0pt;
 
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  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>K152005</span></p>
 
-
  </td>
 
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  <td width=116 valign=top style='width:86.85pt;border-top:none;border-left:
 
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  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
-
 
-
  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>Guelph 08</span></p>
 
-
  </td>
 
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  <td width=213 valign=top style='width:159.55pt;border-top:none;border-left:
 
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  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>(rbs+CrtE) +
 
-
  (rbs+CrtB) + (rbs+CrtI) + (rbs+CrtY) + (rbs+GFP)</span></p>
 
-
  </td>
 
-
  <td width=181 valign=top style='width:136.1pt;border-top:none;border-left:
 
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  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>2 PstI sites in
 
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  CrtI removed.</span></p>
 
-
 
-
  </td>
 
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</tr>
 
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<tr>
 
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  <td width=80 valign=top style='width:60.3pt;border:solid windowtext 1.0pt;
 
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  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>I742158</span></p>
 
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  </td>
 
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  <td width=116 valign=top style='width:86.85pt;border-top:none;border-left:
 
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  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>Edinburgh 07</span></p>
 
-
 
-
  </td>
 
-
  <td width=213 valign=top style='width:159.55pt;border-top:none;border-left:
 
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  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>rbs + CrtZ</span></p>
 
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  </td>
 
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  <td width=181 valign=top style='width:136.1pt;border-top:none;border-left:
 
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  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 
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  padding:0cm 5.4pt 0cm 5.4pt'>
 
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  <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>&nbsp;</span></p>
 
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  </td>
 
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</tr>
 
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</table>
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>&nbsp;</span></p>
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>The above enzymes
 
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all come from <i>Pantoea ananatis</i></span><span lang=EN-GB> (</span><span
 
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lang=EN-GB style='font-family:Arial'>GenBank: D90087.2).</span></p>
 
-
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>&nbsp;</span></p>
 
-
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>However, we have
 
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encountered certain issues during our preliminary research.</span></p>
 
-
 
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<ul style='margin-top:0cm' type=disc>
 
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<li class=MsoNormal><span lang=EN-GB style='font-family:Arial'>In part
 
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    K152005, the “CrtE” sequence (which originated from part K152001) contains
 
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    3 nucleotides different from the NCBI data base.</span></li>
 
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<li class=MsoNormal><span lang=EN-GB style='font-family:Arial'>Originally, the
 
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    registry annotation for K152005 indicated that there was no ribosome
 
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    binding site before gene CrtI (i.e. “…rbs+CrtB+CrtI+rbs+CrtY…”). After
 
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    clarification with Team Guelph 08, we were told that it was solely an
 
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    error in annotation. The registry entry for K152005 has been updated to
 
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    the sequence in the table above.</span></li>
 
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<li class=MsoNormal><span lang=EN-GB style='font-family:Arial'>Teams of
 
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    previous years constructed various composite parts containing two or more
 
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    enzymes of the synthetic pathways (e.g. CrtEBI, CrtEBIY), but they were
 
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    not available in the 2009 Distribution Plates.</span></li>
 
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</ul>
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>&nbsp;</span></p>
 
-
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'> </span><b><u><span
 
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lang=EN-GB style='font-size:18.0pt;font-family:Vivaldi'>Action plan of our team</span></u></b></p>
 
-
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>In our project
 
-
this year, we aim to incorporate the carotenoid biosynthesis pathway in E. coli
 
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and produce different coloured pigments. Potential candidates are lycopene
 
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(red), beta-carotene (orange), and zeaxanthin (yellow). Since these three
 
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compounds are sequential in the biosynthetic pathway, we hope to demonstrate
 
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colour changes from red to orange to yellow, by controlling expression of the
 
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enzymes involved in conversion.</span></p>
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>&nbsp;</span></p>
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>Our action plan
 
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is as follows:</span></p>
 
-
 
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<ol style='margin-top:0cm' start=1 type=1>
 
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<li class=MsoNormal><span lang=EN-GB style='font-family:Arial'>Test the
 
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    activity of K152005 (CrtEBIY).</span></li>
 
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<li class=MsoNormal><span lang=EN-GB style='font-family:Arial'>Construct
 
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    composite biobrick containing CrtE, CrtB and CrtI (i.e. CrtEBI) and test
 
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    its activity.</span></li>
 
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<li class=MsoNormal><span lang=EN-GB style='font-family:Arial'>Transform E.
 
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    coli with CrtEBI and CrtY. Test if activation of CrtY can cause colour
 
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    changes in vivo (by converting red lycopene to orange beta-carotene).</span></li>
 
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<li class=MsoNormal><span lang=EN-GB style='font-family:Arial'>Characterise
 
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    our composite biobricks and the colour output.</span></li>
 
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<li class=MsoNormal><span lang=EN-GB style='font-family:Arial'>Test
 
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    compatibility with biobricks from previous iGEM teams and see if we can
 
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    convert their outputs into pigment production.</span></li>
 
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</ol>
 
-
 
-
<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>&nbsp;</span></p>
 
-
 
-
<p class=MsoNormal style='margin-left:18.0pt;text-indent:-18.0pt'><span
 
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lang=EN-GB style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
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</span></span><b><u><span lang=EN-GB style='font-family:Arial'>Test activity of
 
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K152005</span></u></b></p>
 
-
 
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<p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-GB
 
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style='font-family:Arial'>We wish to test the feasibility of incorporating the
 
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carotenoid biosynthetic pathway into <i>E. coli</i>, which does not produce
 
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carotenoids naturally. K152005 contains the enzymes for beta-carotene
 
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formation, and is a quick way to verify our concept.</span></p>
 
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<p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-GB
 
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style='font-family:Arial'>&nbsp;</span></p>
 
-
 
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<p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-GB
 
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style='font-family:Arial'>We transformed E. coli (TOP10) using K152005 from the
 
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2009 Distribution Plate (Week 2). After incubation for about 3 days, some light
 
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yellowish pigmention was visible. The long incubation time may be due to the
 
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fact that K152005 does not contain promoter. Although only small amount of
 
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yellow pigment was observed (instead of the expected orange colour), this gave
 
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us confidence that the carotenoid biosynthetic pathway was working in <i>E.
 
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coli</i>.</span></p>
 
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<p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-GB
 
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style='font-family:Arial'>&nbsp;</span></p>
 
-
 
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<p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-GB
 
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style='font-family:Arial'>We will then put K152005 under a strong promoter
 
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(R0011, constitutive) to see if stronger colour production can be achieved.</span></p>
 
-
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>&nbsp;</span></p>
 
-
 
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<p class=MsoNormal style='margin-left:18.0pt;text-indent:-18.0pt'><span
 
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lang=EN-GB style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
-
 
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</span></span><b><u><span lang=EN-GB style='font-family:Arial'>Construct
 
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composite Biobrick CrtEBI</span></u></b></p>
 
-
 
-
<p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-GB
 
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style='font-family:Arial'>We wish to produce the first coloured compound in the
 
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pathway, lycopene (red), which can then be converted into other colours by
 
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subsequent enzymatic reaction.</span></p>
 
-
 
-
<p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-GB
 
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style='font-family:Arial'>&nbsp;</span></p>
 
-
 
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<p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-GB
 
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style='font-family:Arial'>In order to “pause” at lycopene, we need enzymes
 
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CrtE, CrtB and CrtI only. Currently in the registry there is no available
 
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Biobrick containing CrtEBI, so we decide to construct our own.</span></p>
 
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<p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-GB
 
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style='font-family:Arial'>&nbsp;</span></p>
 
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<p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-GB
 
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style='font-family:Arial'>After transforming <i>E. coli</i> with K118014
 
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(rbs+CrtE), K118006 (rbs+CrtB) and K118005 (rbs+CrtI), we construct composite
 
-
biobrick by ligating the three genes together.</span></p>
 
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<p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>&nbsp;</span></p>
 
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<p class=MsoNormal style='margin-left:18.0pt;text-indent:-18.0pt'><span
 
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lang=EN-GB style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
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</span></span><b><u><span lang=EN-GB style='font-family:Arial'>Test colour
 
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changes by CrtY</span></u></b></p>
 
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-
<p class=MsoNormal style='margin-left:18.0pt;text-indent:-18.0pt'><span
 
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lang=EN-GB style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
-
</span></span><b><u><span lang=EN-GB style='font-family:Arial'>Characterisation
 
-
of Biobricks and colour output</span></u></b></p>
 
-
 
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<p class=MsoNormal style='margin-left:18.0pt;text-indent:-18.0pt'><span
 
-
lang=EN-GB style='font-family:Symbol'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
-
</span></span><b><u><span lang=EN-GB style='font-family:Arial'>Test
 
-
compatibility with other biobricks.</span></u></b></p>
 
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</div>
 
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</body>
 
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</html>
 

Latest revision as of 16:47, 28 July 2009