Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Oct

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October


  • 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)
        • Backbone pSB1A2_T7 (digested with SpeI, PstI)
          • pSB1A2_T7
        • Insert pSB1C3_alsS_ilvC_ilvD_kivD (digested with XbaI, PstI)
          • alsS_ilvC_ilvD_kivD
      • 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)
          • Backbone pSB1A2_T7 (digested with SpeI, PstI)
            • pSB1A2_T7
          • Insert pSB1C3_alsS_ilvC_ilvD_kivD_adhA (digested with XbaI, PstI)
            • alsS_ilvC_ilvD_kivD_adhA
        • 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