Team:TU-Eindhoven/LEC/Lab

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Revision as of 21:44, 26 September 2012

GECO protein expression and isolation

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.

TODO: COLUMN FILTRATION

Yeast transformation

Yeast transformation.jpg

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.

Transformation chart.png

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 modified yeast transformation protocol.

Device tests

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.

Device test.jpg

Spectrophotometry

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Plasmid construction

Plasmid box.jpg

The following non-BioBrick plasmids have been constructed:

name	vector	inserts	description
pYES2/CT-X-GECO	pYES2/CT	R-GECO1, G-GECO1.1, B-GECO1	High copy-number shuttle vector
pYES3/CT-X-GECO	pYES3/CT	R-GECO1, G-GECO1.1, B-GECO1	High copy-number shuttle vector
pET_28a	X-GECO/pET_28a	R-GECO1, G-GECO1.1, B-GECO1	Expression vector for E. coli

The following plasmids were received from addgene.org:

name	vector	inserts	description
CMV-X-GECO	CMV	R-GECO1, G-GECO1.1, B-GECO1	Shipping vector use by addgene.org, CMV vector was not used in this project

These plasmids were kindly donated by H. Iida & K. Iida:

name	vector	inserts	description
pBCT -CCH1H	pBCT	CCH1H	Low copy-number shuttle vector, encodes the CCH1 protein
pBCT-CCH1H-HA4	pBCT	CCH1H-HA4	Low copy-number shuttle vector, encodes HA4-tagged CCH1
pBCT-CCH1H-EGFP	pBCT	CCH1H-EGFP	Low copy-number shuttle vector, encodes EGFP-tagged CCH1
YCpT-MID1	YCpT	MID1	Low copy-number shuttle vector, encodes the MID1 protein
YCpT-MID1-EGFP	YCpT	MID1-EGFP	Low copy-number shuttle vector, encodes EGFP-tagged MID1

@@ Line 25: / Line 25: @@
 [[File:Plasmid_box.jpg |300px|link=]]
 <p>The following non-BioBrick plasmids have been constructed:</p>
-<html><table><thead><tr>
+<html><div id="groupparts"style="min-height:100px;width:700px;">
-<th>name</th><th>vector</th><th>inserts</th><th>description</th>
+<style>
+    .status_cell { width: 8px; }
+    .status_star { width: 2em; }
+    .c4 { width: 80px; white-space:normal; }
+    .c6 { width: 150px; white-space:normal; }
+    .c7 { width: 50px; white-space:normal; }
+</style>
+<table class="pgrouptable tablesorter" style="width: 100%;" cellpadding="0" cellspacing="0"><thead><tr>
+<th style="width:100px;">name</th><th style="width:80px;">vector</th><th style="width:200px;">inserts</th><th style="width:suto;">description</th>
 </tr></thead>
 <tbody>
 <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>
 <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>
-<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>
+<tr><td>pET<sub>28a</sub></td><td>X-GECO/pET<sub>28a</sub></td><td>R-GECO1, G-GECO1.1, B-GECO1</td><td>Expression vector for E. coli </td></tr>
 </tbody></table></html>
 <p>The following plasmids were received from addgene.org:</p>
-<html><table><thead><tr>
+<html><table class="pgrouptable tablesorter" style="width: 100%;" cellpadding="0" cellspacing="0"><thead><tr>
-<th>name</th><th>vector</th><th>inserts</th><th>description</th>
+<th style="width:65px;">name</th><th style="width:40px;">vector</th><th style="width:175px;">inserts</th><th style="width:auto;">description</th>
 </tr></thead>
 <tbody>
@@ Line 50: / Line 60: @@
 <tr><td>YCpT-MID1</td ><td>YCpT</td><td>MID1</td><td>Low copy-number shuttle vector, encodes the MID1 protein</td></tr>
 <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>
-</tbody></table></html>
+</tbody></table></div></html>
 <h3>References</h3>