"
Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Oct
From 2014.igem.org
(Difference between revisions)
| Line 107: | Line 107: | ||
</ul> | </ul> | ||
| - | <li>For the protein expression analysis of AlsS, IlvC, IlvD and kivD we made a <a href="https://2014.igem.org/https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Cultivation%20for%20Expression%20of%20recombinant%20proteins" target="_blank">cultivation</a>. Samples were taken like explained in the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#FastCellLysisforSDS-PAGE" target="_blank">cell lysis for a SDS-PAGE Protocol</a>. Protein expression was induced when the culture reached a OD<sub>600</sub> 0,8 with rhamnose. The first sample was taken before the induction. Additionalle we took samples | + | <li>For the protein expression analysis of AlsS, IlvC, IlvD and kivD we made a <a href="https://2014.igem.org/https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Cultivation%20for%20Expression%20of%20recombinant%20proteins" target="_blank">cultivation</a>. Samples were taken like explained in the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#FastCellLysisforSDS-PAGE" target="_blank">cell lysis for a SDS-PAGE Protocol</a>. Protein expression was induced when the culture reached a OD<sub>600</sub> 0,8 with rhamnose. The first sample was taken before the induction. Additionalle we took samples two and four, 21 and 23 hours later. Of these samples, we made a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis%20%28SDS-PAGE%29" target="_blank">SDS Page</a> |
| + | |||
| + | <div class="element" style="height:350px; width:600px; text-align:center"> | ||
| + | <a href="https://static.igem.org/mediawiki/2014/a/aa/Bielefeld-CeBiTec_14-10-16_SDS_T7_IB.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/a/aa/Bielefeld-CeBiTec_14-10-16_SDS_T7_IB.jpg" height="230px"></a><br><font size="1">SDS page from pSB1A2_<i>T7</i>_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> </font> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | |||
| + | </li> | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
Revision as of 13:31, 16 October 2014
 |
October |
- pSB1C3_T7_adhA
- This week we tried to reclone pSB1A2_T7_adhA into the pSB1C3 backbone.
- Because the recloning didn't worked well till now, we decided to restrict the used backbone pSB1C3_RFP with EcoRI, XbaI, SpeI and PstI to minimize the chance of religation.
- pSB1C3_T7_adhA we cut with EcoRI and PstI
- Clean up and Ligation as explained in the BioBrick Assembly
- Transformation with electrocompotetent cells
- Colony PCR (rev_pB1C3_adhA, fw_adhA_pSB1C3)
- Annealing temperature: 65 °C
- Bands as expected (~1200 bp)
- Liquid cultures for a restriction digest were prepared.
- Plasmid isolation of pSB1C3_T7_adhA
- Restriction digestion with EcoRI and PstI
- Bands as expected (backbone: ~2,2 kb and insert: ~1,2 kb)
- pSB1A2_T7_alsS_ilvC_ilvD_kivD
- This week we tried to combine the alsS_ilvC_ilvD_kivD construt with the T7 promotor.
- BioBrick Assembly (Suffix)
- Colony PCR (fw_ilvD_kivD, rev_pSB1C3_kiVD)
- Annealing temperature: 65 °C
- Bands as expected (~ 17500 bp)
- Liquid culture for a restriction digest was prepared.
- Plasmid isolation of pSB1A2-T7_alsS_ilvC_ilvD_kivD
- Restriction digestion with EcoRI and PstI
- Bands as expected (backbone: ~2,2 kb and insert: ~7500 kb)
- For the protein expression analysis of AlsS, IlvC, IlvD and kivD we made a cultivation. Samples were taken like explained in the cell lysis for a SDS-PAGE Protocol. Protein expression was induced when the culture reached a OD600 0,8 with rhamnose. The first sample was taken before the induction. Additionalle we took samples two and four, 21 and 23 hours later. Of these samples, we made a SDS Page
SDS page from pSB1A2_T7_alsS_ilvC_ilvD_kivD - pSB1A2_T7_alsS_ilvC_ilvD_kivD_adhA
- This week we tried to combine the alsS_ilvC_ilvD_kivD_adhA construt with the T7 promotor.
- BioBrick Assembly (Suffix)
- Colony PCR (fw_ilvD_kivD, rev_pSB1C3_kiVD)
- Annealing temperature: 65 °C
- Bands as expected (~ 17500 bp)
- Liquid culture for a restriction digest was prepared.
- Plasmid isolation of pSB1A2-T7_alsS_ilvC_ilvD_kivD_adhA
- Restriction digestion with EcoRI and PstI
- Bands as expected (backbone: ~2,2 kb and insert: ~8500 kb)
- For the protein expression analysis of AlsS, IlvC, IlvD, KivD and AdhA we made a cultivation. Samples were taken like explained in the cell lysis for a SDS-PAGE Protocol. Protein expression was induced when the culture reached a OD600 0,8 with rhamnose. The first sample was taken before the induction. Additionalle we took samples one, two and three and 20 hours later. Of these samples, we made a SDS Page
