Team:Cambridge-JIC/Constructs/progress template

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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. Tubes: CpI 1, CpI 11
    • Backbone Fragment 2 (35s - Hyg promoter): B14, P16, P14. Tubes: CpI 2
    • N7 fragment: B15, P13, P22. Tubes: CpI 3
    • eforRed gene(cytoplasmic): B13, P1, P2. Tubes: CpI 4
    • eforRed gene(nuclear): B13, P1, P3. Tubes: CpI 5
    • amilCP gene(nuclear): B10, P4, P5. Tubes: CpI 6
    • tsPurple gene(cytoplasmic): B12, P6, P7. Tubes: CpI 7
    • tsPurple gene(nuclear): B12, P6, P8. Tubes: CpI 8
    • asPink gene(nuclear): B9, P9, P10. CpI 9
    • aeBlue gene(nuclear): B11, P11, P12. Tubes: CpI 10

      • After running the gel, the concentrations of CpI 1, CpI 2 and CpI 3 were much lower compared to the chromoprotein genes (see gel picture). Amplification of CpI 3 failed. The problem was an alteration to the primers necessary for amplification. Re-ran PCR for CpI 3. Modification: Appropriate primers (P13 & P22) and the extension phase of PCR has been reduced from 2 min to 15 sec since N7 is only ~300bp long.

    PCR Excell Protocol

    Gel Picture


Attempt 2:

    Date: 21.07.2014

    UIDs of primers/plasmids used:

    • Backbone Fragment 1(nosT - Hyg promoter): B14, P15, P17. Tubes: CpII 1
    • Backbone Fragment 2 (35s - Hyg promoter): B14, P16, P14. Tubes: CpII 2
    • N7 fragment: B15, P13, P22. Tubes: CpII 3
    • eforRed gene(cytoplasmic): B13, P1, P2. Tubes: CpII 4
    • eforRed gene(nuclear): B13, P1, P3. Tubes: CpII 5
    • amilCP gene(nuclear): B10, P4, P5. Tubes: CpII 6
    • tsPurple gene(cytoplasmic): B12, P6, P7. Tubes: CpII 7
    • tsPurple gene(nuclear): B12, P6, P8. Tubes: CpII 8
    • asPink gene(nuclear): B9, P9, P10. CpII 9
    • aeBlue gene(nuclear): B11, P11, P12. Tubes: CpII 10

    PCR Excell Protocol

    Gel Picture

Gibson

Attempt 1:
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E-Coli transformation

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Agrobacteria transformation

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Spore preparation

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Spore transformation

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Evaluation

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