Difference between revisions of "Team:Bielefeld-CeBiTec/Notebook/PlasmidDisplay"
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<p> | <p> | ||
<ul> | <ul> | ||
| − | <li>Purification of | + | <li>Purification of his-tagged lacI</li> |
<li>Test digest of transformed Biobricks: all clones as expected!</li> | <li>Test digest of transformed Biobricks: all clones as expected!</li> | ||
| − | <li> | + | <li>Plasmid isolation of BBa_R0010 and BBa_K592008 </li> |
<ul> | <ul> | ||
</ul> | </ul> | ||
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<div class="panel-body"> | <div class="panel-body"> | ||
| − | <p>. | + | <p> |
| + | <ul> | ||
| + | <li>Verification of lacI-His </li> | ||
| + | <ul> | ||
| + | <li>SDS-PAGE for analyzing the purifity of purified lacI-His</li> | ||
| + | <li>lacI-His was expected in the 38 kDa bands </li> | ||
| + | <li>Bands (size 38 kDa) were cut out and destained. | ||
| + | </ul> | ||
| + | </ul></p> | ||
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| − | <p>...</p> | + | <p> |
| + | <ul> | ||
| + | <li>Identification of the bands cut before</li> | ||
| + | <ul> | ||
| + | <li>Tryptic digestion of the proteins from the bands. </li> | ||
| + | <li>Measurement of the digested proteins via MALDI-TOF/TOF.</li> | ||
| + | <li>The measured peptide spectre was compared with spectre of other proteins with an <i>E. coli</i> database. </li> | ||
| + | <li>LacI identified (Mascot score: 765).</li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | </p> | ||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 21:27, 20 August 2015
Plasmid Repressor Interaction Assay
- Heat shock transformation of Biobricks needed for further assays was performed. Glycerol stocks were generated and the plasmids isolated according to the kits manual. The relevant Biobricks were:
- BBa_K525998: T7 promotor and RBS
- BBa_C0012: lacI repressor with LVA Tag
- BBa_R0010: lac Promotor
- BBa_K592008: lac Operator
- Furthermore a plasmid with a gene coding for a histagged lacI Protein was transformed, which was kindly provided by Stefan Hoffmann.
- Purification of his-tagged lacI
- Test digest of transformed Biobricks: all clones as expected!
- Plasmid isolation of BBa_R0010 and BBa_K592008
- Verification of lacI-His
- SDS-PAGE for analyzing the purifity of purified lacI-His
- lacI-His was expected in the 38 kDa bands
- Bands (size 38 kDa) were cut out and destained.
- Identification of the bands cut before
- Tryptic digestion of the proteins from the bands.
- Measurement of the digested proteins via MALDI-TOF/TOF.
- The measured peptide spectre was compared with spectre of other proteins with an E. coli database.
- LacI identified (Mascot score: 765).