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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Oct
From 2014.igem.org
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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 (~ 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 one, two and three and 20 hours later. Of these samples, we made a SDS Page
- 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
