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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Oct
From 2014.igem.org
(Difference between revisions)
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</ul></ul> | </ul></ul> | ||
</ul> </ul> | </ul> </ul> | ||
| + | <br> | ||
| + | <ul> | ||
| + | <li><b><i>pSB1A2_T7_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></i></b></li> | ||
| + | <ul> | ||
| + | <li>This week we tried to combine the <i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></i> construt with the <i>T7</i> 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 pSB1A2_T7(digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" 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 pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>(digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" 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>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | |||
| + | </ul> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvD_kivD" target="_blank">fw_ilvD_kivD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_kivD" target="_blank">rev_pSB1C3_kiVD</a>) | ||
| + | </li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 65 °C</li> | ||
| + | <li>Bands as expected (~ 17500 bp)</li> | ||
| + | </ul> | ||
| + | <li>Liquid culture for a restriction digest was prepared.</li> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1A2-<i>T7</i>_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></li> | ||
| + | <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#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a></li> | ||
| + | <ul> | ||
| + | <li>Bands as expected (backbone: ~2,2 kb and insert: ~7500 kb)</li> | ||
| + | </ul> | ||
| + | <li>Successful sequencing</li> | ||
| + | <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 one, two and three and 20 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></li> | ||
| + | </ul> | ||
| + | </ul> | ||
<br> | <br> | ||
Revision as of 13:01, 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)
- Successful sequencing
- 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 one, two and three and 20 hours later. Of these samples, we made a SDS Page
