Team:Cambridge-JIC/Constructs/progress template

From 2014.igem.org

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<li> 35s - asPink - N7 - nosT</li>
<li> 35s - asPink - N7 - nosT</li>
<li> 35s - aeBlue - N7 - nosT</li>
<li> 35s - aeBlue - N7 - nosT</li>
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<br></br>
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<br></br>By expressing the chromoproteins in the pGreen vector with the 35s promoter and the nosT terminaor, we control for the possiblity of the promoter/terminator not functioning in our chassis.
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By expressing the chromoproteins in the pGreen vector with the 35s promoter and the nosT terminaor, we control for the possiblity of the promoter/terminator not functioning in our chassis.
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The N7 fragment allows for the selected constructs to localise chromoprotein to the nucleus. It is believed by Bernardo Pollak that this may end up increasing the likelihood of detecting the chromoproteins with the naked eye.
The N7 fragment allows for the selected constructs to localise chromoprotein to the nucleus. It is believed by Bernardo Pollak that this may end up increasing the likelihood of detecting the chromoproteins with the naked eye.
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Attempt 1:
Attempt 1:
<ul>
<ul>
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<li>Start date/time:</li>
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<p>Date: 18.07.2014</p>
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<li>End date/time:</li>
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<p>UIDs of primers/plasmids used:</p>
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<li> UIDs of primers/plasmids used:  
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<ul><li>Backbone Fragment 1(nosT - Hyg promoter): B14, P15, P17 </li>
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<ul>
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<li>Backbone Fragment 2 (35s - Hyg promoter): B14, P16, P14</li>
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<li> A2, P12, P13</li>
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<li>N7 fragment: B15, P13, P22</li>
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<li> B3, P14, P13</li>
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<li>eforRed gene(cytoplasmic): B13, P1, P2</li>
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<li> A4, P12, P13 </ul></li>
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<li> eforRed gene(nuclear): B13, P1, P3</li>
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<li> amilCP gene(nuclear): B10, P4, P5</li>
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<li> tsPurple gene(cytoplasmic): B12, P6, P7</li>
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<li> tsPurple gene(nuclear): B12, P6, P8</li>
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<li> asPink gene(nuclear): B9, P9, P10</li>
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<li> aeBlue gene(nuclear): B11, P11, P12</li></ul>
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<li><a href="http://pcrspreadsheet.com">PCR Spreadsheet (on google drive)</a></li>
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<li><a href="http://google.com/GEL_27_07_2014">Gel 27/07/2014</a></li>
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</ul>
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<p><a href="http://pcrspreadsheet.com">PCR Spreadsheet (on google drive)</a></p>
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<p><a href="http://google.com/GEL_27_07_2014">Gel 27/07/2014</a></p>
</div>
</div>

Revision as of 14:51, 24 July 2014

Chromoprotein Constructs

Design

Aim of the construct

    Chromoproteins represent ideal reporter genes. Unlike fluorescent protein, from which chromoproteins were originally derived, they require no specialised optical equipment for detection. Our aim is to use a selection of such proteins, donated by the iGEM 2012 Uppsala team, as our most basic and main output in regards to the Marchantia framework.

    We decided to test the following chromoprotein constructs:

  • 35s - eforRed - nosT
  • 35s - eforRed - N7 - nosT
  • 35s - amilCP - N7 - nosT
  • 35s - tsPurple - nosT
  • 35s - tsPurple - N7 - nosT
  • 35s - asPink - N7 - nosT
  • 35s - aeBlue - N7 - nosT


    • By expressing the chromoproteins in the pGreen vector with the 35s promoter and the nosT terminaor, we control for the possiblity of the promoter/terminator not functioning in our chassis. The N7 fragment allows for the selected constructs to localise chromoprotein to the nucleus. It is believed by Bernardo Pollak that this may end up increasing the likelihood of detecting the chromoproteins with the naked eye.

End goal plasmid

  • insert image here

Experimentation (up to date)

PCR

Attempt 1:

    Date: 18.07.2014

    UIDs of primers/plasmids used:

    • Backbone Fragment 1(nosT - Hyg promoter): B14, P15, P17
    • Backbone Fragment 2 (35s - Hyg promoter): B14, P16, P14
    • N7 fragment: B15, P13, P22
    • eforRed gene(cytoplasmic): B13, P1, P2
    • eforRed gene(nuclear): B13, P1, P3
    • amilCP gene(nuclear): B10, P4, P5
    • tsPurple gene(cytoplasmic): B12, P6, P7
    • tsPurple gene(nuclear): B12, P6, P8
    • asPink gene(nuclear): B9, P9, P10
    • aeBlue gene(nuclear): B11, P11, P12

    PCR Spreadsheet (on google drive)

    Gel 27/07/2014

Gibson

Attempt 1:
  • Start date/time:
  • End date/time:
  • Comments:

E-Coli transformation

Attempt 1:
  • Start date/time:
  • End date/time:
  • Comments:

Agrobacteria transformation

Attempt 1:
  • Start date/time:
  • End date/time:
  • Induce comments:
  • Growth comments:
  • Electroporation/selection comments:

Spore preparation

Attempt 1:
  • Start date/time:
  • End date/time:
  • Comments:

Spore transformation

Attempt 1:
  • Start date/time:
  • End date/time:
  • Comments:

Evaluation

  • Start date/time:
  • End date/time:
  • Comments:

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