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

From 2014.igem.org


September

Week 1     09/01 - 09/07
Week 1     09/01 - 09/07
  • T7_alsS_ilvC_ilvD_kivD
    • This week we tried to verify the positive cutures that were identified last week by a restriction digest
  • pSB1K3_alsS_ilvC_ilcD_kivD_adhA

Week 2     09/08 - 09/14
Week 2     09/08 - 09/14

Week 3     09/15 - 09/21
Week 3     09/15 - 09/21
  • pSB1C3_alsS_ilvC_ilvD_kivD_adhA
    • This week we tried to contruct the pSB1C3_alsS_ilvC_ilvD_kivD_adhA-Plasmid by NEB BioBrick Assembly
      • NEB BioBrick Assembly
        • Upstream part pSB1K3_alsS_ilvC_ilvD_kivD (digested with EcoRI, SpeI)
          • alsS_ilvC_ilvD_kivD
        • Downstream part pSB1K3_adhA(digested with XbaI, PstI)
          • adhA
        • Destination part pSB1C3_RFP (digested with EcoRI, PstI)
          • alsS_ilvC_ilvD_kivD
      • Until now we didn't purified Inserts out of the gel if the backbone had another antibiotic resistence. We were using the PCR purification. But because it didn't worked out, we analyzed all our cut samples by gelelectrophorese. Thereby we discovered that the pSB1K3_alsS_ilvC_ilvD_kivD has an illigal restriction side.
        Because of the integration of RBS's between all genes in the alsS_ilvC_ilvD_kivD-Plasmid, a restriction side between alsS and a RBS occurs.
        New primers were ordered: rv_ilvC_alsS-new and fw_alsS_ilvC-new
Week 4     09/22 - 09/28
Week 4     09/22 - 09/28
Week 5     09/29 - 10/05
Week 5 09/29 - 10/05
  • pSB1C3_ptac_alsS_ilvC_ilcD_kivD
    • This week we tried to combine the pSB1C3_alsS_ilvC_ilvD_kivD contruct (without illegale restriction side) with the ptac promotor.
    • BioBrick Assembly (Suffix and Prefix)
      • Backbone (digested with SpeI, PstI)
        • pSB1C3_ptac
      • Insert (digested with XbaI, PstI)
        • alsS_ilvC_ilvD_kivD
        AND:
      • Backbone (digested with EcoRI, XbaI)
        • alsS_ilvC_ilvD_kivD_pSB1C3
      • Insert (digested with EcoRI, SpeI)
        • ptac
    • Transformation with electrocompotetent cells
    • Colony PCR (VF-Primer, rv_ilvC_alsS-new)
      • Annealing temperature: 55°C
      • Bands as expected (~ 3000 bp)
    • Liquid culture for a restriction digest was prepared.
    • Plasmid isolation of pSB1C3-ptac_alsS_ilvC_ilvD_kivD
    • Restriction digestion with XbaI and PstI
      • Bands as expected (backbone: ~ 2200 bp and insert: ~ 9500 bp)
    • Successful sequencing
  • pSB1C3_alsS_ilvC_ilcD_kivD_adhA
    • This week we tried to combine the pSB1C3_alsS_ilvC_ilvD_kivD contruct (without illegale restriction side) with the adhA from L. lactis.
    • BioBrick Assembly (Suffix and Prefix)
      • Backbone (digested with SpeI, SpeI)
        • pSB1C3_adhA
      • Insert (digested with EcoRI, PstI)
        • alsS_ilvC_ilvD_kivD
        AND:
      • Backbone (digested with SpeI, PstI)
        • pSB1C3_alsS_ilvC_ilvD_kivD
      • Insert (digested with XbaI, PstI)
        • ptac
    • Transformation with electrocompotetent cells
    • Colony PCR (fw_ilvD_kivD, rev_pSB1C3_adhA)
      • Annealing temperature: 55°C
      • Bands as expected (~ 3000 bp)
    • Liquid culture for a restriction digest was prepared.
    • Plasmid isolation of pSB1C3_alsS_ilvC_ilvD_kivD_adhA
    • Restriction digestion with EcoRI and PstI
      • Bands as expected (backbone: ~ 2200 bp and insert: ~ 9500 bp)
    • Successful sequencing
  • pSB1A2_T7_adhA
    • This week we tried to contruct the pSB1A2_T7_adhA-Plasmid
      • Colony PCR (fw_pSB1C3_adhA, rev_pSB1C3_adhA)
        • Annealing temperature: 65 °C
        • Bands as expected (~ 1200 bp)
      • Liquid culture for a restriction digest was prepared.
      • Plasmid isolation of pSB1A2-T7_adhA
      • Restriction digestion with EcoRI and PstI
        • Bands as expected (backbone: ~ 2200 bp and insert: ~ 1300 bp)
      • Successful sequencing
      • For the protein expression analysis of 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

        SDS page from pSB1A2_T7_adhA
        The sizes of the AdhA is ~ 35 Da
    • GC-MS analysis of the isobutanol production
      • For the analysis of the isobutanol production we decided to use a GC-MS analysis. Because we will cultivate in LB medium, we made a calibration line with LB and the following concentrations of isobutanol for further analysis and quantification of our samples.
        • 0,5 % isobutanol
        • 0,1 % isobutanol
        • 0,05 % isobutanol
        • 0,01 % isobutanol
        • 0,001 % isobutanol
        Additionally we prepared GC-grade aceton with 1% 2-Butanol as an internal standard. >br>The samples were treated as described below:
        • Solvent extraction with 100µl LB-sample and 900µl aceton
        • 5 min centrifugation
        • 150 µl supernatant in the GC-MS vials for the analysis
      • The detection of isobutanol works for all tested concentrations but the 2-butanol peak was to high, so we need to do this again with 0.1% 2-Butanol.

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