Team:TU-Eindhoven/LEC/Lab

From 2012.igem.org

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<h3>Expected very soon!</h3>
 
<h3>Plasmid construction</h3>
<h3>Plasmid construction</h3>
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[[File:Plasmid_box.jpg|right|250px|link=]]
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[[File:Plasmid_box.jpg|left|300px|link=]]
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<p>
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The following plasmids have been constructed:
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<ul>
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<li>pYES2/R-GECO: High copy-number shuttle vector  with R-GECO1 for expression in yeast</li>
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<li>pYES2/G-GECO: High copy-number shuttle vector  with G-GECO1.1 for expression in yeast</li>
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<li> pYES2/B-GECO: High copy-number shuttle vector  with B-GECO1 for expression in yeast</li>
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<li>pYES3/R-GECO: High copy-number shuttle vector  with R-GECO1 for expression in yeast</li>
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<li>pYES3/G-GECO: High copy-number shuttle vector  with G-GECO1.1 for expression in yeast</li>
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<li> pYES3/B-GECO: High copy-number shuttle vector  with B-GECO1 for expression in yeast</li>
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<li>pET<sub>28a</sub>/R-GECO: Vector for expression of R-GECO1 in E. coli</li>
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<li>pET<sub>28a</sub>/G-GECO: Vector for expression of G-GECO1.1 in E. coli</li>
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<li>pET<sub>28a</sub>/B-GECO: Vector for expression of B-GECO1 in E. coli</li>
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<li>pSB1C3/R-GECO: Standard BioBrick plasmid with R-GECO1</li>
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<li>pSB1C3/G-GECO: Standard BioBrick plasmid with G-GECO1.1</li>
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<li>pSB1C3/B-GECO: Standard BioBrick plasmid with B-GECO1</li>
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</ul>
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The following plasmids were received from addgene.org:
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<ul>
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<li>CMV/R-GECO: Encodes R-GECO1, CMV vector not used in this project</li>
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<li>CMV/G-GECO: Encodes G-GECO1.1, CMV vector not used in this project</li>
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<li>CMV/B-GECO: Encodes B-GECO1, CMV vector not used in this project</li>
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</ul>
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These plasmids were kindly donated by H. Iida & K. Iida:
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<ul>
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<li>pBCT/CCH1H: Low copy-number shuttle vector, encodes the CCH1 protein</li>
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<li>pBCT/CCH1H-HA4: Low copy-number shuttle vector, encodes HA4-tagged CCH1 </li>
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<li>pBCT/CCH1H-EGFP: Low copy-number shuttle vector, encodes EGFP-tagged CCH1</li>
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<li>YCpT-MID1: Low copy-number shuttle vector, encodes the MID1 protein</li>
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<li>YCpT-MID1-EGFP: Low copy-number shuttle vector, encodes EGFP-tagged MID1</li>
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</ul>
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</p>
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<h3>GECO protein expression and isolation</h3>
<h3>GECO protein expression and isolation</h3>
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[[File:GECO_in_BL21.jpg|right|300px|link=]]
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[[File:GECO_in_BL21.jpg|right|150px|link=]]
<p>The three GECOs have been expressed in E. coli BL21 to yield a large amount of the proteins for characterization. Cells were cultured in large Erlenmeyer flasks. The results were visually impressive: After lysis the flasks turned vibrantly red and green respectively. The blue GECO however looked much the same as the green GECO.</p>
<p>The three GECOs have been expressed in E. coli BL21 to yield a large amount of the proteins for characterization. Cells were cultured in large Erlenmeyer flasks. The results were visually impressive: After lysis the flasks turned vibrantly red and green respectively. The blue GECO however looked much the same as the green GECO.</p>
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<p>A weekly item on our schedule was yeast transformation. It takes about a week to complete a transformation, that is, to add one plasmid to an existing strain variant. Because success is not guaranteed we choose to introduce plasmids in various orders in parallel. This resulted in many variants that we assigned a unique number for convenience. The same number was used on plates, cultures and cryostocks. In total 49 variants were made.</p>
<p>A weekly item on our schedule was yeast transformation. It takes about a week to complete a transformation, that is, to add one plasmid to an existing strain variant. Because success is not guaranteed we choose to introduce plasmids in various orders in parallel. This resulted in many variants that we assigned a unique number for convenience. The same number was used on plates, cultures and cryostocks. In total 49 variants were made.</p>
[[File:Transformation chart.png|right|300px|link=]]
[[File:Transformation chart.png|right|300px|link=]]
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<p>Initially, yeast transformations failed or yielded only several colonies, a lot less than expected. A review of transformation protocols showed that the shock protocol we used should be more efficient than electroporation or other common methods. We tried to transform another yeast strain with our plasmids which yielded the expected high transformation efficiency. Unfortunately that strain was not compatible with the auxotrophic markers on the plasmids we wanted to introduce. Our problem was remedied by using more plasmid DNA for the transformation, as described in our [[Team:TU-Eindhoven/Protocols|modified yeast transformation protocol]].
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<p>Initially, yeast transformations failed or yielded only several colonies, a lot less than expected. A review of transformation protocols showed that the shock protocol we used should be more efficient than electroporation or other common methods. We tried to transform another yeast strain with our plasmids which yielded the expected high transformation efficiency. Unfortunately that strain was not compatible with the auxotrophic markers on the plasmids we wanted to introduce. Our problem was remedied by using more plasmid DNA for the transformation, as described in our [[Team:TU-Eindhoven/Protocols|modified yeast transformation protocol]].</p>
<h3>Device tests</h3>
<h3>Device tests</h3>
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<p>The device was tested with yeast containing all three plasmids, thus over expressing channels and expressing GECOs. A quick inspection with the naked eye did not show any sign of visual light coming off the device, not even in a dark room. We think that the yeast was not concentrated enough to show a clear response.</p>
[[File:Device test.jpg|right|300px|link=]]
[[File:Device test.jpg|right|300px|link=]]
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<h3>Spectrophotometry</h3>
<h3>Spectrophotometry</h3>
[[File:Spectra yeast.png|right|300px|link=]]
[[File:Spectra yeast.png|right|300px|link=]]
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<h3></h3>
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<p>
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The following non-BioBrick plasmids have been constructed:
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<html><table><thead><tr>
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<th>name</th><th>vector</th><th>inserts</th><th>description</th>
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</tr></thead>
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<tbody>
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<tr><td>pYES2/CT-X-GECO</td><td>pYES2/CT</td><td>R-GECO1, G-GECO1.1, B-GECO1</td><td>High copy-number shuttle vector </td></tr>
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<tr><td>pYES3/CT-X-GECO</td><td>pYES3/CT</td><td>R-GECO1, G-GECO1.1, B-GECO1</td><td>High copy-number shuttle vector </td></tr>
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<tr><td>pET<sub>28a</sub></td><td>X-GECO</td><td>pET<sub>28a</sub></td><td>R-GECO1, G-GECO1.1, B-GECO1</td><td>Expression vector for E. coli </td></tr>
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</tbody></table></html>
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The following plasmids were received from addgene.org:
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<table><thead><tr>
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<th>name</th><th>vector</th><th>inserts</th><th>description</th>
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</tr></thead>
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<tbody>
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<tr><td>CMV </td><td>R-GECO1, G-GECO1.1, B-GECO1</td><td>Shipping vector use by addgene.org, CMV vector was not used in this project</td></tr>
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</tbody></table></html>
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These plasmids were kindly donated by H. Iida & K. Iida:
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<html><table><thead><tr>
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<th>name</th><th>vector</th><th>inserts</th><th>description</th>
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</tr></thead>
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<tbody>
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<tr><td>pBCT -CCH1H</td><td>pBCT </td><td>CCH1H</td><td>Low copy-number shuttle vector, encodes the CCH1 protein</td></tr>
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<tr><td>pBCT-CCH1H-HA4</td><td>pBCT</td><td>CCH1H-HA4</td><td>Low copy-number shuttle vector, encodes HA4-tagged CCH1</td></tr>
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<tr>< td>pBCT-CCH1H-EGFP</td><td>pBCT</td><td>CCH1H-EGFP</td><td>Low copy-number shuttle vector, encodes EGFP-tagged CCH1</td></tr>
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<tr><td>YCpT-MID1</td ><td>YCpT</td><td>MID1</td><td>Low copy-number shuttle vector, encodes the MID1 protein</td></tr>
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<tr><td>YCpT-MID1-EGFP</td> <td>YCpT</td><td>MID1-EGFP</td><td>Low copy-number shuttle vector, encodes EGFP-tagged MID1</td></tr>
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</tbody></table></html>
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</p>
<h3>References</h3>
<h3>References</h3>

Revision as of 21:09, 26 September 2012

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