Team:Cambridge-JIC/Constructs/progress template

From 2014.igem.org

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<html>
<html>
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<h1> Template Construct Name</h1>
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<h1> Chromoprotein Constructs</h1>
<h2> Design </h2>
<h2> Design </h2>
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<h4> Aim of the construct </h4>
<h4> Aim of the construct </h4>
<ul>  
<ul>  
-
<li> Describe aims here </li>
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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.
 +
<br></br>
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We decided to test the following chromoprotein constructs:
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<br></br>
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<li> 35s - eforRed - nosT</li>
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<li> 35s - eforRed - N7 - nosT</li>
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<li> 35s - amilCP - N7 - nosT</li>
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<li> 35s - tsPurple - nosT</li>
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<li> 35s - tsPurple - N7 - nosT</li>
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<li> 35s - asPink - N7 - nosT</li>
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<li> 35s - aeBlue - N7 - nosT</li>
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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.
 +
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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</ul>
</ul>
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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 (35s - Hyg promoter): B14, P16, P14. Tubes: CpI 1</li>
 +
<li>Backbone Fragment 2(Hyg promoter - nosT): B14, P15, P17. Tubes: CpI 2</li>
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<li>N7 fragment: B15, P13, P22. Tubes: CpI 3</li>
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<li>eforRed gene(cytoplasmic): B13, P1, P2. Tubes: CpI 4</li>
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<li> eforRed gene(nuclear): B13, P1, P3. Tubes: CpI 5</li>
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<li> amilCP gene(nuclear): B10, P4, P5. Tubes: CpI 6</li>
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<li> tsPurple gene(cytoplasmic): B12, P6, P7. Tubes: CpI 7</li>
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<li> tsPurple gene(nuclear): B12, P6, P8. Tubes: CpI 8</li>
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<li> asPink gene(nuclear): B9, P9, P10. CpI 9</li>
 +
<li> aeBlue gene(nuclear): B11, P11, P12. Tubes: CpI 10</li></ul>
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<p>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.
 +
The DNA was then purified using the QIAGEN QiaQuick Gel Purification Kit. The DNA was eluted in 50 ul of EB buffer. Due to the large elution volume and the lower yield of the backbone DNA, the concentrations (measured using Nanodrop) were lower than the suggested 10ng/ul threshold for Gibson.
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</p></ul>
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<p><a href="http://pcrspreadsheet.com">PCR Excell Protocol</a></p>
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<p><a href="http://google.com/GEL_27_07_2014">Gel Picture</a></p>
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</div>
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<br>
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Attempt 2:
<ul>
<ul>
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<li> A2, P12, P13</li>
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<p>Date: 21.07.2014</p>
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<li> B3, P14, P13</li>
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<p>UIDs of primers/plasmids used:</p>
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<li> A4, P12, P13 </ul></li>
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<ul><li>Backbone Fragment 1 (35s - Hyg promoter): B14, P16, P14. Tubes: CpII 1</li>
 +
<li>Backbone Fragment 2(Hyg promoter - nosT): B14, P15, P17. Tubes: CpII 2</li>
 +
<li>N7 fragment: B15, P13, P22. Tubes: CpII 3</li>
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<li>eforRed gene(cytoplasmic): B13, P1, P2. Tubes: CpII 4</li>
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<li> eforRed gene(nuclear): B13, P1, P3. Tubes: CpII 5</li>
 +
<li> amilCP gene(nuclear): B10, P4, P5. Tubes: CpII 6</li>
 +
<li> tsPurple gene(cytoplasmic): B12, P6, P7. Tubes: CpII 7</li>
 +
<li> tsPurple gene(nuclear): B12, P6, P8. Tubes: CpII 8</li>
 +
<li> asPink gene(nuclear): B9, P9, P10. CpII 9</li>
 +
<li> aeBlue gene(nuclear): B11, P11, P12. Tubes: CpII 10</li></ul>
 +
<p>Due to the low yield of the DNA purification fro Attempt 1, the entire PCR was repeated. Upon gel separation we realised the PCRs were nearly identical (see Gel Picture), which is what we were expecting. However, the DNA was euted into 10ul of EB buffer instead of 50ul during the gel purification step, which increased the concentrations of CpII 4-10 significantly. However, CpII 1-3 concentrations were still below the 10 ng DNA/ul range. </p></ul>
 +
<p><a href="http://pcrspreadsheet.com">PCR Excell Protocol</a></p>
 +
<p><a href="http://google.com/GEL_27_07_2014">Gel Picture</a></p>
 +
</div>
-
<li><a href="http://pcrspreadsheet.com">PCR Spreadsheet (on google drive)</a></li>
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Attempt 3:
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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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</ul>
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<p>Date: 22.07.2014</p>
 +
<p>UIDs of primers/plasmids used:</p>
 +
<ul><li>Backbone Fragment 1 (35s - Hyg promoter): B14, P16, P14. Tubes: CpIII 1</li>
 +
<li>Backbone Fragment 2 - Full Size(Hyg promoter - nosT): B14, P15, P17. Tubes: CpIII 2 RBLB</li>
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<li>Backbone Fragment 2 - LB(Hyg promoter - KanR): B14, P18, P17. Tubes: CpIII 2 LB</li>
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<li>Backbone Fragment 2 - RB(KanR - nosT): B14, P15, P19. Tubes: CpIII 2 RB</li>
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<li>N7 fragment: B15, P13, P22. Tubes: CpIII 3</li>
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<p>The backbone fragments were re-aplified to allow for future assemblies to be made. The PCR was altered to run for 35 amplification cycles instead of 30, which increase the yield of DNA. </p></ul>
 +
<p><a href="http://pcrspreadsheet.com">PCR Excell Protocol</a></p>
 +
<p><a href="http://google.com/GEL_27_07_2014">Gel Picture</a></p>
</div>
</div>
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Attempt 1:
Attempt 1:
<ul>
<ul>
-
<li>Start date/time:</li>
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<li>Date: 21.07.2014</li>
<li>End date/time:</li>
<li>End date/time:</li>
<li>Comments:</li>
<li>Comments:</li>

Latest revision as of 17:32, 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 (35s - Hyg promoter): B14, P16, P14. Tubes: CpI 1
    • Backbone Fragment 2(Hyg promoter - nosT): B14, P15, P17. 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. The DNA was then purified using the QIAGEN QiaQuick Gel Purification Kit. The DNA was eluted in 50 ul of EB buffer. Due to the large elution volume and the lower yield of the backbone DNA, the concentrations (measured using Nanodrop) were lower than the suggested 10ng/ul threshold for Gibson.

PCR Excell Protocol

Gel Picture


Attempt 2:

    Date: 21.07.2014

    UIDs of primers/plasmids used:

    • Backbone Fragment 1 (35s - Hyg promoter): B14, P16, P14. Tubes: CpII 1
    • Backbone Fragment 2(Hyg promoter - nosT): B14, P15, P17. 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

    Due to the low yield of the DNA purification fro Attempt 1, the entire PCR was repeated. Upon gel separation we realised the PCRs were nearly identical (see Gel Picture), which is what we were expecting. However, the DNA was euted into 10ul of EB buffer instead of 50ul during the gel purification step, which increased the concentrations of CpII 4-10 significantly. However, CpII 1-3 concentrations were still below the 10 ng DNA/ul range.

PCR Excell Protocol

Gel Picture

Attempt 3:

Gibson

Attempt 1:

E-Coli transformation

Attempt 1:

Agrobacteria transformation

Attempt 1:

Spore preparation

Attempt 1:

Spore transformation

Attempt 1:

Evaluation

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