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| - | <title>Colour pigment: Carotenoid</title>
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| - | <div class=Section1>
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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'> </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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| - |
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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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| - |
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| - | <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'> </span></p>
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| - |
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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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| - |
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| - | <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'> </span></p>
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| - |
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| - | [[[Image:Image001.jpg]]]
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| - |
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| - | <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'> </span></p>
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| - |
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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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| - |
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| - | <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'> </span></p>
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| - |
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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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| - |
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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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| - |
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| - | <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'> </span></p>
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| - |
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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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| - |
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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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| - | padding:0cm 5.4pt 0cm 5.4pt'>
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| - |
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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 + 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'> </span></p>
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| - | </td>
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| - |
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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>
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| - | </td>
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| - |
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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'> </span></p>
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| - | </td>
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| - | </tr>
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| - | <tr>
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| - |
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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>
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| - |
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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'>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>
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| - |
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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 + 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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| - |
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| - | <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'> </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>
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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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| - | padding:0cm 5.4pt 0cm 5.4pt'>
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| - |
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| - | <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>Guelph 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+CrtE) +
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| - | (rbs+CrtB) + (rbs+CrtI) + (rbs+CrtY) + (rbs+GFP)</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'>2 PstI sites in
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| - | CrtI removed.</span></p>
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| - |
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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'>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>
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| - |
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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 + 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'> </span></p>
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| - | </td>
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| - | </tr>
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| - |
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| - | </table>
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| - |
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| - | <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'> </span></p>
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| - |
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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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| - |
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| - | <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'> </span></p>
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| - |
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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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| - |
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| - | <ul style='margin-top:0cm' type=disc>
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| - |
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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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| - |
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| - | <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'> </span></p>
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| - |
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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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| - |
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| - | <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'>In our project
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| - | 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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| - |
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| - | <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'> </span></p>
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| - |
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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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| - |
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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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| - |
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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>
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| - |
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| - | <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'> </span></p>
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| - |
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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"'>
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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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| - |
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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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| - |
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| - | <p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-GB
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| - | style='font-family:Arial'> </span></p>
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| - |
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| - | <p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-GB
| |
| - | style='font-family:Arial'>We transformed E. coli (TOP10) using K152005 from the
| |
| - | 2009 Distribution Plate (Week 2). After incubation for about 3 days, some light
| |
| - | 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
| |
| - | us confidence that the carotenoid biosynthetic pathway was working in <i>E.
| |
| - | coli</i>.</span></p>
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| - |
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| - | <p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-GB
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| - | style='font-family:Arial'> </span></p>
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| - |
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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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| - |
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| - | <p class=MsoNormal><span lang=EN-GB style='font-family:Arial'> </span></p>
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| - |
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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"'>
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| - |
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| - | </span></span><b><u><span lang=EN-GB style='font-family:Arial'>Construct
| |
| - | composite Biobrick CrtEBI</span></u></b></p>
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| - |
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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 produce the first coloured compound in the
| |
| - | pathway, lycopene (red), which can then be converted into other colours by
| |
| - | subsequent enzymatic reaction.</span></p>
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| - |
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| - | <p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-GB
| |
| - | style='font-family:Arial'> </span></p>
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| - |
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| - | <p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-GB
| |
| - | style='font-family:Arial'>In order to “pause” at lycopene, we need enzymes
| |
| - | CrtE, CrtB and CrtI only. Currently in the registry there is no available
| |
| - | Biobrick containing CrtEBI, so we decide to construct our own.</span></p>
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| - |
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| - | <p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-GB
| |
| - | style='font-family:Arial'> </span></p>
| |
| - |
| |
| - | <p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-GB
| |
| - | style='font-family:Arial'>After transforming <i>E. coli</i> with K118014
| |
| - | (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'> </span></p>
| |
| - |
| |
| - | <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"'>
| |
| - | </span></span><b><u><span lang=EN-GB style='font-family:Arial'>Test colour
| |
| - | changes by CrtY</span></u></b></p>
| |
| - |
| |
| - | <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"'>
| |
| - | </span></span><b><u><span lang=EN-GB style='font-family:Arial'>Characterisation
| |
| - | of Biobricks and colour output</span></u></b></p>
| |
| - |
| |
| - | <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"'>
| |
| - | </span></span><b><u><span lang=EN-GB style='font-family:Arial'>Test
| |
| - | compatibility with other biobricks.</span></u></b></p>
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | </body>
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| - |
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| - | </html>
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