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Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Sep
From 2014.igem.org
(Difference between revisions)
| Line 62: | Line 62: | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
| - | </ul> | + | </ul> <!-- /csoS1-4 --> |
<br> | <br> | ||
| Line 87: | Line 87: | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
| - | + | <ul> | |
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_csoS1-4_GFP</li> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Nod</i>I</a></li> | ||
| + | <ul> | ||
| + | <li>Bands (as expected (~2000 bp and ~2400 bp)</li> | ||
| + | </ul> | ||
| + | </ul> | ||
</ul> | </ul> | ||
| - | </ul> | + | </ul> <!-- /csoS1-4 and GFP --> |
<br> | <br> | ||
| + | |||
| + | <ul> <!-- csoS1-4_GFP Fusion mit T7 --> | ||
| + | <li><b><i>csoS1-4_GFP</i> and T7</b></li> | ||
| + | <ul> | ||
| + | <li>We tried to assemble our pSB1C3_csoS1-4_GFP construct with the T7 promotor.</li> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li> | ||
| + | <ul> | ||
| + | <li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li> | ||
| + | <ul> | ||
| + | <li>pSB1A2_T7</li> | ||
| + | </ul> | ||
| + | <li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li> | ||
| + | <ul> | ||
| + | <li><i>csoS1-4_GFP</i></li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | |||
| + | |||
| + | </ul> | ||
| + | </ul> <!-- /csoS1-4_GFP Fusion --> | ||
<ul> <!-- csoS1-4 Fusion mit can --> | <ul> <!-- csoS1-4 Fusion mit can --> | ||
Revision as of 20:37, 29 September 2014
 |
September |
 |
- csoS1-4
- We tried to amplify csoS1-4 (shell proteins) for a fusion with GFP.
- PCR amplification (fw-GFP-csoS1B, rv-rv-GFP-csoS1A)
- Annealing temperature: 54 °C
- Bands as expected (~4000 bp (~1800 bp csoS1-4, ~2200 bp pSB1C3))
- PCR products were purified
- csoS1-4 and GFP
- We tried to assemble the shell proteins of the carboxysome and GFP.
- Gibson Assembly with pSB1C3_csoS1-4 and GFP
- Transformation with electrocompotetent cells
- Colony PCR (fw-csoS1A-GFP, rv-csoS1B-GF)
- Annealing temperature: 68 °C
- Bands as expected (~1000 bp)
- Plasmid isolation of pSB1C3_csoS1-4_GFP
- Restriction digestion with NodI
- Bands (as expected (~2000 bp and ~2400 bp)
- csoS1-4_GFP and T7
- We tried to assemble our pSB1C3_csoS1-4_GFP construct with the T7 promotor.
- BioBrick Assembly (Suffix)
- can and csoS1-4
- This week we tried to find positive clones of our transformation.
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~3600 bp)
- Plasmid isolation of pSB1C3_can_csoS1-4
- Restriction digestion with EcoRI and PstI
- Bands as expected (~2100 bp and ~3300 bp)
- Hneap and T7
- This week we tried to assemble the T7 promotor with the Hneap.
- BioBrick Assembly (Suffix)
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~2300 bp)
