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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Oct
From 2014.igem.org
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| - | + | <li><b>Restriction digestion as a control</b></li> | |
| - | + | <div class="element" style="height:350px; width:220px; text-align:center"> | |
| + | <a href="https://static.igem.org/mediawiki/2014/b/b4/Bielefeld-CeBiTec_14_17_10_Kontrollverdau_10_7.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/b/b4/Bielefeld-CeBiTec_14_17_10_Kontrollverdau_10_7.jpg" height="230px"></a><br><font size="1">Agarose gel control restriction digestion from several constructs with the restriction. IB stands for <i>pSB1C3_alsS_ilvC_ilvD_kivD</i>. As Ladder we used a 1kb Ladder</a>. </font></ul> | ||
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Revision as of 01:19, 18 October 2014
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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 (~ 7500 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
The sizes of the included proteins are ~61 Da for the AlsS,
~54 Da for the IlvC, ~65 Da for the IlvD and ~62 Da 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 two, four, six, 22 and 24 hours later. Of these samples, we made a SDS Page
SDS page from pSB1A2_T7_alsS_ilvC_ilvD_kivD_adhA
The sizes of the included proteins are ~61 Da for the AlsS, ~54 Da for the IlvC,
~65 Da for the IlvD, ~62 Da for KivD and ~36 Da for AdhA - Restriction digestion as a control
Agarose gel control restriction digestion from several constructs with the restriction. IB stands for pSB1C3_alsS_ilvC_ilvD_kivD. As Ladder we used a 1kb Ladder.
